Methods for identifying compounds for regulating muscle mass or function using corticotropin releasing factor receptors

ABSTRACT

Screening methods for identifying compounds that bind to or activate corticotropin releasing factor 2  receptors (CRF 2 R) and regulate or potentially regulate skeletal muscle mass or function in vivo. Also disclosed are screening methods for identifying compounds that prolong or augment the activation of CRF 2 Rs or of CRF 2 R signal transduction pathways, increase CRF 2 R or increase CRF expression are provided. Pharmaceutical compositions comprising CRF 2 R agonists, antibodies to CRF 2 R and methods for increasing skeletal muscle mass or function or for the treatment of skeletal muscle atrophy using CRF 2 R as the target for intervention and methods for treatment of muscular dystrophies are described.

TECHNICAL FIELD

[0001] The present invention relates to methods of identifying candidatecompounds for regulating skeletal muscle mass or function or regulatingthe activity or expression of a corticotropin releasing factor-2receptor (CRF₂R). The invention also relates to methods for thetreatment of skeletal muscle atrophy or methods for inducing skeletalmuscle hypertrophy using CRF₂R as the target for intervention and tomethods of treating muscular dystrophies using CRF₂R and corticotropinreleasing factor-1 receptor (CRF₁R) as targets.

BACKGROUND

[0002] CRFR and Ligands

[0003] There are two corticotropin releasing factor receptors,identified to date (CRF₁R and CRF₂R) which belong to G-protein coupledreceptor (GPCR) class. Agonist activation of CRF₁R or CRF₂R leads toG_(αs) activation of adenylate cyclase. Adenylate cyclase catalyzes theformation of cAMP, which in turn has multiple effects including theactivation of protein kinase A, intracellular calcium release andactivation of mitogen-activated protein kinase (MAP kinase). In otherstudies, the enhancement of intracellular inositol triphosphatesynthesis, after agonist activation of CRF receptors, suggests thatCRFRs also couple to G_(αq).

[0004] CRF₁R and CRF₂R have been cloned from human, rat, mouse, chicken,cow, catfish, frog and sheep. CRF₁R and CRF₂R each have a uniquedistribution patterns. In humans three isoforms, alpha, beta and gamma,of the CRF₂R receptor have been cloned. Homologs for alpha and betaCRF₂R have been identified in rat.

[0005] Several ligands/agonists of the CRFRs are known. Corticotropinreleasing factor (or hormone, CRF or CRH) binds to and activates CRF₁Rand CRF₂R. CRF is a major modulator of the body's responses to stress.This 41-amino acid peptide presides over a panoply of neuronal,endocrine, and immune processes as the primary regulator of thehypothalamus-pituitary-adrenal hormonal axis (HPA axis). In addition,there is substantial sequence homology between CRF and the amphibianpeptide sauvagine as well as the telostian peptide urotensin, both ofwhich act as agonists of CRF₁R and CRF₂R. These three peptides havesimilar biological properties as hypotensive agents and ACTHsecretogogues. In addition, a mammalian congener of urotensin,urocortin, has been characterized.

[0006] The CRF receptors can be distinguished, from non-CRFRs,pharmacologically through the use of receptor selective agonists andantagonists. These selective agonists and antagonist, along with theCRFR knockout mice, have been useful in determining which CRF receptormediates specific biological responses.

[0007] The role of CRF₁R has been fairly well established. Mice in whichthe CRF₁R gene has been ablated (CRF₁R knockout) demonstrate an impairedstress response and reduced anxiety-like behavior. CRF₁R is a majormediator of the HPA axis. Specifically, corticotropin releasing factor,which is released from the hypothalamus and transported to the anteriorpituitary via the hypothalamic-hypophysial portal system, interacts withthe CRF₁R present on cells located in the anterior pituitary. Agonistactivation of the CRF₁R results in release of ACTH from the cells of theanterior pituitary into the systemic circulation. The released ACTHbinds the ACTH receptor present on cells located in the adrenal cortex,resulting in the release of adrenal hormones including corticosteroids.Corticosteroids mediate many effects including, but not limited to,immune system suppression via a mechanism which involves thymic andsplenic atrophy. Thus activation of the CRF₁R indirectly results in thedown-regulation of the immune system via activation of the HPA axis.

[0008] The role of CRF₂R is less well developed. Mice in which the CRF₂Rgene has been ablated (CRF₂R knockout) demonstrate an impaired foodintake reduction following stimulation with urocortin, lack ofvasodilation, but a normal stress response. Experiments with CRF₂Rdemonstrated that CRF₂R is responsible for the hypotensive/vasodilatoryeffects of CRFR agonists and for the reduction in food intake observedfollowing treatment of mice with CRFR agonists.

[0009] Skeletal Muscle Atrophy and Hypertrophy

[0010] Skeletal muscle is a plastic tissue which readily adapts tochanges in either physiological demand for work or metabolic need.Hypertrophy refers to an increase in skeletal muscle mass while skeletalmuscle atrophy refers to a decrease in skeletal muscle mass. Acuteskeletal muscle atrophy is traceable to a variety of causes including,but not limited to: disuse due to surgery, bed rest, or broken bones;denervation/nerve damage due to spinal cord injury, autoimmune disease,or infectious disease; glucocorticoid use for unrelated conditions;sepsis due to infection or other causes; nutrient limitation due toillness or starvation; and space travel. Skeletal muscle atrophy occursthrough normal biological processes, however, in certain medicalsituations this normal biological process results in a debilitatinglevel of muscle atrophy. For example, acute skeletal muscle atrophypresents a significant limitation in the rehabilitation of patients fromimmobilizations, including, but not limited to, those accompanying anorthopedic procedure. In such cases, the rehabilitation period requiredto reverse the skeletal muscle atrophy is often far longer than theperiod of time required to repair the original injury. Such acute disuseatrophy is a particular problem in the elderly, who may already sufferfrom substantial age-related deficits in muscle function and mass,because such atrophy can lead to permanent disability and prematuremortality.

[0011] Skeletal muscle atrophy can also result from chronic conditionssuch as cancer cachexia, chronic inflammation, AIDS cachexia, chronicobstructive pulmonary disease (COPD), congestive heart failure, geneticdisorders, e.g., muscular dystrophies, neurodegenerative diseases andsarcopenia (age associated muscle loss). In these chronic conditions,skeletal muscle atrophy can lead to premature loss of mobility, therebyadding to the disease-related morbidity.

[0012] Little is known regarding the molecular processes which controlatrophy or hypertrophy of skeletal muscle. While the initiating triggerof the skeletal muscle atrophy is different for the various atrophyinitiating events, several common biochemical changes occur in theaffected skeletal muscle fiber, including a decrease in proteinsynthesis and an increase in protein degradation and changes in bothcontractile and metabolic enzyme protein isozymes characteristic of aslow (highly oxidative metabolism/slow contractile protein isoforms) tofast (highly glycolytic metabolism/fast contractile protein isoforms)fiber switch. Additional changes in skeletal muscle which occur includethe loss of vasculature and remodeling of the extracellular matrix. Bothfast and slow twitch muscle demonstrate atrophy under the appropriateconditions, with the relative muscle loss depending on the specificatrophy stimuli or condition. Importantly, all these changes arecoordinately regulated and are switched on or off depending on changesin physiological and metabolic need.

[0013] The processes by which atrophy and hypertrophy occur areconserved across mammalian species. Multiple studies have demonstratedthat the same basic molecular, cellular, and physiological processesoccur during atrophy in both rodents and humans. Thus, rodent models ofskeletal muscle atrophy have been successfully utilized to understandand predict human atrophy responses. For example, atrophy induced by avariety of means in both rodents and humans results in similar changesin muscle anatomy, cross-sectional area, function, fiber type switching,contractile protein expression, and histology. In addition, severalagents have been demonstrated to regulate skeletal muscle atrophy inboth rodents and in humans. These agents include anabolic steroids,growth hormone, insulin-like growth factor I, and beta adrenergicagonists. Together, these data demonstrate that skeletal muscle atrophyresults from common mechanisms in both rodents and humans.

[0014] While some agents have been shown to regulate skeletal muscleatrophy and are approved for use in humans for this indication, theseagents have undesirable side effects such as hypertrophy of cardiacmuscle, neoplasia, hirsutism, androgenization of females, increasedmorbidity and mortality, liver damage, hypoglycemia, musculoskeletalpain, increased tissue turgor, tachycardia, and edema. Currently, thereare no highly effective and selective treatments for either acute orchronic skeletal muscle atrophy. Thus, there is a need to identify othertherapeutic agents which regulate skeletal muscle atrophy.

[0015] Muscular Dystrophies

[0016] Muscular dystrophies encompass a group of inherited, progressivemuscle disorders, distinguished clinically by the selective distributionof skeletal muscle weakness. The two most common forms of muscledystrophy are Duchenne and Becker dystrophies, each resulting from theinheritance of a mutation in the dystrophin gene, which is located atthe Xp21 locus. Other dystrophies include, but are not limited to,limb-girdle muscular dystrophy which results from mutation of multiplegenetic loci including the p94 calpain, adhalin, γ-sarcoglycan, andβ-sarcoglycan loci; fascioscapulohumeral (Landouzy-Dejerine) musculardystrophy, myotonic dystrophy, and Emery-Dreifuss muscular dystrophy.The symptoms of Duchenne muscular dystrophy, which occurs almostexclusively in males, include a waddling gait, toe walking, lordosis,frequent falls and difficulty in standing up and climbing stairs.Symptoms start at about 3-7 years of age with most patients confined toa wheelchair by 10-12 years and many die at about 20 years of age due torespiratory complications. Current treatment for Duchenne musculardystrophy includes administration of prednisone (a corticosteroid drug),which while not curative, slows the decline of muscle strength anddelays disability. Corticosteroids, such as prednisone, are believed toact by blocking the immune cell activation and infiltration which areprecipitated by muscle fiber damage resulting from the disease.Unfortunately, corticosteroid treatment also results in skeletal muscleatrophy which negates some of the potential benefit of blocking theimmune response in these patients. Thus, there is a need to identifytherapeutic agents which slow the muscle fiber damage and delay theonset of disability in patients with muscular dystrophies, but cause alesser degree of skeletal muscle atrophy than current therapies.

[0017] One problem associated with identification of compounds for usein the treatment of skeletal muscle atrophy or of muscular dystrophieshas been the lack of good screening methods for the identification ofsuch compounds. Applicants have now found that CRF₂Rs are involved inthe regulation of skeletal muscle mass or function and that agonists ofCRF₂Rs are able to block skeletal muscle atrophy and/or inducehypertrophy of skeletal muscle. The present invention solves the problemof identifying compounds for the treatment of muscle atrophy byproviding screening methods using CRF₂R which can be used to identifycandidate compounds useful for the treatment of muscle atrophy. Thepresent invention also solves the problem of finding compounds fortreatment of muscle dystrophies by providing a screening method toidentify candidate compounds which activate both the CRF₁R and CRF₂R.

SUMMARY OF THE INVENTION

[0018] The present invention relates to the use of CRFRs to identifycandidate compounds that are potentially useful in the treatment ofskeletal muscle atrophy and or to induce skeletal muscle hypertrophy. Inparticular, the invention provides in vitro methods for identifyingcandidate compounds for regulating skeletal muscle mass or functioncomprising contacting a test compound with a cell expressing CRF₂R, orcontacting a test compound with isolated CRF₂R, and determining whetherthe test compound either binds to or activates the CRF₂R. Anotherembodiment of the invention relates to a method for identifyingcandidate therapeutic compounds from a group of one or more candidatecompounds which have been determined to bind to or activate CRF₂Rcomprising administering the candidate compound to a non-human animaland determining whether the candidate compound regulates skeletal musclemass or muscle function in the treated animal. A further embodiment ofthe invention relates to a method for identifying candidate compoundsfor regulating skeletal muscle mass or function comprising, in anyorder: (i) contacting a test compound with a cell expressing afunctional CRF₂R, and determining a level of activation of CRF₂Rresulting from the test compound; (ii) contacting a test compound with acell expressing a functional CRF₁R, and determining the level ofactivation of CRF₁R resulting from the test compound; followed by (iiii)comparing the level of CRF₂R activation and the level of CRF₁Ractivation; and (iv) identifying those test compounds that show similaractivity toward CRF₂R and CRF₁R or show selectivity for CRF₂R ascandidate compounds for regulating skeletal muscle mass or function.

[0019] The invention further provides methods for identifying candidatecompounds that prolong or augment the agonist-induced activation ofCRF₂R or of a CRF₂R signal transduction pathway. These methods comprisein any order or concurrently: (i) contacting a test compound; with acell which expresses functional CRF₂R (ii) treating the cell with aCRF₂R agonist for a sufficient time and at a sufficient concentration tocause desensitization of the CRF₂R in control cells; followed by (iii)determining the level of activation of CRF₂R and identifying testcompounds that prolong or augment the activation of a CRFR or a CRFRsignal transduction pathway as candidate compounds for regulatingskeletal muscle mass or function. In a particular embodiment, thepresent invention relates to a method of identifying candidatetherapeutic compounds from a group of one or more candidate compoundsdetermined to prolong or augment the activation of a CRF₂R or of a CRF₂Rsignal transduction pathway comprising: administering the candidatecompound, in conjunction with a CRF₂R agonist, to a non-human animal anddetermining whether the candidate compound regulates skeletal musclemass or function in the treated animal.

[0020] The invention further provides methods for identifying candidatecompounds that increase CRF₂R expression comprising contacting a testcompound with a cell or cell lysate containing a reporter geneoperatively associated with a CRF ₂R gene regulatory element anddetecting expression of the reporter gene. Test compounds that increaseexpression of the reporter gene are identified as candidate compoundsfor increasing CRF₂R expression. In a particular embodiment, the presentinvention relates to a method of determining whether those candidatecompounds which increase CRF₂R expression can be used to regulateskeletal muscle mass or function in vivo by administering a candidatecompound to a non-human animal and determining whether the candidatecompound regulates skeletal muscle mass or function in the treatedanimal.

[0021] The invention further provides methods for identifying candidatecompounds that increase CRF expression comprising contacting a testcompound with a cell or cell lysate containing a reporter geneoperatively associated with a CRF gene regulatory element and detectingexpression of the reporter gene. Test compounds that increase expressionof the reporter gene are identified as candidate compounds forincreasing CRF expression. In a particular embodiment, the presentinvention relates to a method of determining whether those candidatecompounds which increase CRF expression can be used to regulate skeletalmuscle mass or function in vivo by administering a candidate compound toa non-human animal and determining whether the candidate compoundregulates skeletal muscle mass or function in the treated animal.

[0022] The present invention also relates to the use of CRF₂R agonists,expression vectors encoding a functional CRF₂R, expression vectorsencoding a constitutively active CRF₂R or compounds that increaseexpression of CRF₂R, or CRF to treat skeletal muscle atrophy. Inparticular, the invention provides methods of treating skeletal muscleatrophy, in a subject in need of such treatment, comprisingadministering to the subject a safe and effective amount of a CRF₂Ragonist, an expression vector encoding a functional CRF₂R, an expressionvector encoding a constitutively active CRF₂R, an expression vectorencoding a CRF or CRF analog, or a compound that increases expression ofCRF₂R, or CRF. In a particular embodiment, the present invention relatesto a method for treating skeletal muscle atrophy in a subject in need ofsuch treatment comprising administering to the subject a safe andeffective amount of a CRF₂R agonist in conjunction with a safe andeffective amount of a compound that prolongs or augments theagonist-induced activation of CRF₂R, or of a CRF₂R signal transductionpathway.

[0023] The present invention also relates to the use of a CRF₂R agonistto increase skeletal muscle mass or function in a subject. Inparticular, the invention provides methods of increasing skeletal musclemass or function in a subject in which such an increase is desirable,comprising identifying a subject in which an increase in muscle mass orfunction is desirable and administering to the subject a safe andeffective amount of a CRFR agonist.

[0024] The invention further provides for pharmaceutical compositionscomprising a safe and effective amount of a CRF₂R agonist and apharmaceutically-acceptable carrier. In a particular embodiment thepharmaceutical composition comprises a chimeric or human antibodyspecific for a CRF₂R. In another particular embodiment thepharmaceutical composition comprises a CRF or CRF analog, preferablyurocortin II.

[0025] The present invention also provides for antibodies to CRF₂R andin particular to chimeric or human antibodies that are agonists ofCRF₂R.

[0026] Throughout this application various publications are referenced.The disclosures of these publications in their entireties are herebyincorporated by reference in this application in order to more fullydescribe the state of the art to which this invention pertains.

SEQUENCE LISTING DESCRIPTION

[0027] Each of the CRFR nucleotide and protein sequences or CRF analogprotein sequence included in the sequence listing, along with thecorresponding Genbank or Derwent accession number(s) and animal speciesfrom which it is cloned, is shown in Table I. Also shown are accessionnumbers for related nucleotide sequences that encode identical, ornearly identical, amino acid sequences as the sequence shown in thesequence listing. These related sequences differ mainly in the amount of5′ or 3′ untranslated sequence shown. TABLE I Genbank (GB) or SEQ ID NO:Derwent (D) Sequence nucleotide, Accession No. for Related Genbank (GB)or description amino acid Species nucleotide sequence Derwent (D)Accession Nos. CRF₁R 1, 2 Homo sapiens X72304 (GB) E11431 (GB) L23332(GB) I92584 (D) T37068 (D) Q81952 (D) CRF₁R 3, 4 Homo sapiens L23333(GB) variant CRF₁R 5, 6 Homo sapiens NM_004382 (GB) variant CRF₁R 7, 8Homo sapiens AF180301 (GB) variant CRF₂R alpha 9, 10 Homo sapiens U34587(GB) E12752 (GB) T12247 (D) NM_001883 (GB) T66508 (D) CRF₂R beta 11, 12Homo sapiens AF011406 (GB) CRF₂R 13, 14 Homo sapiens AF019381 (GB) gammaCRF₁R 15, 16 Rattus T28970 (D) L25438 (GB) norvegicus L24096 (GB) I92586(D) Q81954 (D) AH006791 (GB) CRF₂R alpha 17, 18 Rattus U16253 (GB)NM_022714 (GB) norvegicus X01009 (D) T12243 (D) CRF₂R beta 19, 20 RattusT12244 (D) variant norvegicus CRF₁R 21, 22 Mus musculus NM_007762 (GB)X72305 (D) CRF₂R 23, 24 Mus musculus T28972(D) U17858 (GB) CRF₂R 25, 26Mus musculus NM_009953 (GB) CRF₁R 27, 28 Ovis aries AF054582 (GB) CRF₁R29, 30 Xenopus laevis Y14036 (GB) CRF₂R 31, 32 Xenopus laevis Y14037(GB) CRF₁R 33, 34 Ameiurus AF229359 (GB) nebulosus CRF₁R 35, 36 AmeiurusAF229361 (GB) nebulosus CRF₂R 37, 38 Ameiurus AF229360 (GB) nebulosusCRF₁R 39, 40 Bos taurus AB055434 (GB) CRF₁R 41, 42 Gallus gallus L41563(GB) Urocortin II 43 Mus musculus AF331517 Urocortin- 44 Homo sapiensBC002647 related peptide

BRIEF DESCRIPTION OF THE FIGURES

[0028]FIG. 1 demonstrates the anti-atrophy effect of the CRF₁R/CRF₂Ragonist, sauvagine (administered subcutaneously, 2× daily), on themedial gastrocnemius muscle in the mouse sciatic nerve denervationatrophy model.

[0029]FIG. 2 demonstrates the anti-atrophy effect of sauvagine(administered continuously by osmotic minipump) on the tibialis anteriormuscle in the mouse sciatic nerve denervation atrophy model.

[0030]FIGS. 3A and 3B demonstrate the anti-atrophy effect of sauvagine(administered continuously by osmotic minipump) onglucocorticoid-induced atrophy of the tibialis anterior muscle (FIG. 3A)and the medial gastrocnemius muscle (FIG. 3B).

[0031]FIG. 4A demonstrates the anti-atrophy effect of sauvagine(administered subcutaneously, 2× daily) on the casting-induced atrophyof the tibialis anterior muscle and hypertrophy-inducing effect on thenon-casted (normal) tibialis anterior muscle. FIG. 4B demonstrates theanti-atrophy effect of sauvagine on the casting-induced atrophy of themedial gastrocnemius muscle and the hypertrophy inducing effect ofsauvagine on the non-casted (normal) medial gastrocnemius muscle.

[0032]FIG. 5 demonstrates the anti-atrophy and hypertrophy inducingeffects of sauvagine and urocortin (administered continuously by osmoticminipump) on the tibialis anterior muscle in the mouse sciatic nervedenervation-induced atrophy model.

[0033]FIGS. 6A and 6B demonstrate the anti-atrophy effects of urocortin(administered subcutaneously, 2× daily) on the disuse-induced atrophy ofthe tibialis anterior muscle (FIG. 6A) and of the medial gastrocnemiusmuscle (FIG. 6B).

[0034]FIG. 7 demonstrates in the anti-atrophy effect of sauvagine(administered subcutaneously, 2× daily), in the adrenalectomized ratsciatic nerve denervation-induced atrophy model, on thedenervation-induced atrophy of the tibialis anterior (FIG. 7A), extensordigitorum longus (EDL) (FIG. 7B), soleus (FIG. 7C), medial gastrocnemius(FIG. 7D), and plantaris (FIG. 7E) muscles. In addition, sauvagineinduced hypertrophy of the non-denervated EDL muscle (FIG. 7B).

[0035]FIG. 8 demonstrates that in the mouse sciatic nerve denervationatrophy model, sauvagine (administered continuously by osmotic minipump)had an anti-atrophy effect on the tibialis anterior muscle in wild-typemice but not in CRF₂R knockout mice.

[0036]FIGS. 9A and B demonstrate that in a mouse leg casting disuseatrophy model, sauvagine had an anti-atrophy effect on the EDL andsoleus muscle as measured by mass (FIG. 9A) or muscle function (FIG.9B).

DETAILED DESCRIPTION OF THE INVENTION

[0037] I. Terms and Definitions:

[0038] The following is a list of definitions for terms used herein.

[0039] “Agonist” means any compound, including, but not limited to,antibodies, that activates a receptor. For example, CRFR agonistsinclude, but are not limited to, CRF and CRF analogs.

[0040] “Allelic variant” means a variant form of a given gene or geneproduct. One of skill in the art recognizes that a large number of genesare present in two or more allelic forms in a population and some geneshave numerous alleles.

[0041] “Antibody”, in its various grammatical forms, meansimmunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain an antigenbinding site which specifically binds an antigen. “Purified antibody”means an antibody which has been partially or completely separated fromthe proteins and naturally-occurring organic molecules with which it isnaturally associated. Preferably, the preparation is at least 60%antibody, more preferably at least 75% antibody, more preferably atleast 90% antibody, and most preferably at least 99%, by dry weight,antibody.

[0042] “Binding affinity” means the propensity for a ligand to interactwith a receptor and is inversely related to the dissociation constantfor a specific CRF ligand-CRFR interaction. The dissociation constantcan be measured directly via standard saturation, competition, orkinetics binding techniques or indirectly via pharmacological techniquesinvolving functional assays and endpoints.

[0043] “Chimeric antibody” means an antibody that contains structuralelements from two or more different antibody molecules, i.e., fromdifferent animal species. Chimeric antibodies include, but are notlimited to, antibodies known as “humanized antibodies” which include,but are not limited to, chimeric antibodies generated by the techniqueknown as complementarity determining region grafting.

[0044] “CRF” means corticotropin releasing factor which is the same ascorticotropin releasing hormone (CRH). Exemplary CRF peptides includer/h CRF and ovine CRF (see U.S. Pat. No. 4,415,558), and the like.

[0045] “CRF analog” means substances which act as ligands of CRFRs.Suitable CRF analogs can be obtained from a variety of vertebratespecies and include, but are not limited to, substances such assauvagine (see, e.g., U.S. Pat. No. 4,605,642), urotensin (see, e.g.,U.S. Pat. Nos. 4,908,352; and 4,533,654), mouse urocortin II (SEQ ID NO:43), human urocortin-related peptide (SEQ ID NO:44) (Reyes, T. M. etal., Proc. Nat'l Acad Sci 98:2843-2848 (2001)), urocortin (see, e.g., WO97/00063) and the CRF analogs described in U.S. Pat. Nos. 4,415,558;4,489,163; 4,594,329; 4,605,642; 5,109,111; 5,235,036; 5,278,146;5,439,885; 5,493,006; 5,663,292; 5,824,771; 5,844,074; and 5,869,450.Each of which is incorporated herein by reference. Preferred CRF analogsare sauvagine, urocortin, urocortin-related peptide, urocortin-II andurotensin.

[0046] “CRFR agonist” means a compound or molecule which has the abilityto activate CRF₁R or CRF₂R, or both. Activation of CRFRs can be measuredas described hereinafter.

[0047] “CRFR” means CRF ₁R or CRF ₂R.

[0048] “CRF ₁R” means any isoforms of CRF ₁R from any animal species.The CRF ₁R has previously been referred to as CRF-RA, PC-CRF, CRF,(Perrin, M. H., et al. Endocrinology 133:3058-3061 (1993), Chen, R., etal. Proc. Natl. Acad. Sci. USA 90:8967-8971 (1993), Chang, C-P. et al.,Neuron 11:1187-1195 (1993), Kishimoto, T., et al., Proc. Natl. Acad.Sci. USA, 92:1108-1112 (1995) and, Vita, N. et al., FEBS Lett. 335: 1-5(1993)) or the CRH receptor.

[0049] The definition of CRF ₁R includes, but is not limited to, thosereceptors for which the cDNA or genomic sequence encoding the receptorhas been deposited in a sequence database. These sequences includeAccession Nos.: X72304, E11431, L23332, I92584, T37068, T28968, Q81952,L23333, NM_(—)004382, AF180301, T28970, L25438, L24096, I92586, Q81954,AH006791, NM_(—)007762, X72305, AF054582, Y14036, AF229359, AF229361,AB055434 and L41563. The nucleotide and protein sequences of thesereceptors are available from GenBank or Derwent and for conveniencerepresentative sequences are given in the sequence listing herein.

[0050] “CRF ₂R” means any isoform of CRF ₂R from any animal species. CRF₂R has also been referred to as HM-CRF, CRF-RB , (Kishimoto, T., et al.,Proc. Natl. Acad. Sci. USA, 92:1108-1112 (1995) and Perrin, M. et al.Proc. Natl. Acad. Sci. USA 92:2969-2973 (1995)).

[0051] The definition of CRF ₂R receptor includes, but is not limitedto, those receptors for which the DNA sequence encoding the receptor hasbeen deposited in a sequence database. These sequences include AccessionNos.: U34587, E12752, NM_(—)001883, T12247, T66508, AF011406, AF019381,U16253, T12244, T28972, U17858, NM_(—)009953, Y14037 and AF229360. Thenucleotide and protein sequences of these receptors are available fromGenBank or Derwent and for convenience, representative sequences aregiven in the sequence listing herein.

[0052] The term “CRFR” also includes truncated and/or mutated proteinswherein regions of the receptor molecule not required for ligand bindingor signaling have been deleted or modified. For example one of skill inthe art will recognize that a CRFR with one or more conservative changesin the primary amino acid sequence would be useful in the presentinvention. It is known in the art that substitution of certain aminoacids with different amino acids with similar structure or properties(conservative substitutions) can result in a silent change, i.e., achange that does not significantly alter function. Conservativesubstitutes are well known in the art. For example, it is known thatGPCRs can tolerate substitutions of amino acid residues in thetransmembrane alpha-helices, which are oriented toward lipid, with otherhydrophobic amino acids, and remain functional. CRF₁Rs differing from anaturally occurring sequence by truncations and/or mutations such asconservative amino acid substitutions are also included in thedefinition of CRF₁R. CRF₂R differing from a naturally occurring sequenceby truncations and/or mutations such as conservative amino acidsubstitutions are also included in the definition of CRFR₂.

[0053] One of skill in the art would also recognize that CRFRs from aspecies other than those listed above, particularly mammalian species,would be useful in the present invention. One of skill in the art wouldfurther recognize that by using probes from the known CRFR species'sequences, cDNA or genomic sequences homologous to the known sequencecould be obtained from the same or alternate species by known cloningmethods. Such CRF₁R are also included in the definition of CRF₁R andsuch CRF₂R are also included in the definition of CRF₂R.

[0054] In addition, one of skill in the art would recognize thatfunctional allelic variants or functional splice variants of CRFRs mightbe present in a particular species and that these variants would haveutility in the present invention. Splice variants of CRFRs are known,for example U.S. Pat. Nos. 5,888,811; 5,786,203; and 5,728,545, each ofwhich is incorporated herein by reference. Such CRF₁R variants are alsoincluded in the definition of CRF₁R and such CRF₂R variants are alsoincluded in the definition of CRF₂R.

[0055] Fusions of a CRF₁R or CRF₂R polypeptide, or a CRF₁R or CRF₂Rpolypeptide fragment to a non-CRFR polypeptide are referred to as CRFRfusion proteins. Using known methods, one of skill in the art would beable to make fusion proteins of a CRF₁R or a CRF₂R that, while differentfrom native CRF₁R and CRF₂R, would remain useful in the presentinvention. For example the non-CRFR polypeptide may be a signal (orleader) polypeptide sequence which co-translationally orpost-translationally directs transfer of the protein from its site ofsynthesis to another site (e.g., the yeast α-factor leader). Or thenon-CRFR polypeptide may be added to facilitate purification oridentification of the CRFR (e.g., poly-His, or Flag peptide). CRF₁Rfusion proteins are also included within the definition of CRF₁R andCRF₂R fusion proteins are also included within the definition of CRF₂R.

[0056] “CRF₂R signal transduction pathway” means any signaling pathway(e.g., cAMP, MAP kinase) or combination of signaling pathways that aremodulated by the binding of endogenous or exogenous ligands to CRF₂R.

[0057] “Functional CRFRs” refers to CRFRs, which bind CRF or a CRFanalog in vivo or in vitro and are activated as a result of ligandbinding.

[0058] “Fusion gene” means two or more DNA coding sequences operablyassociated so as to encode one hybrid protein. A “fusion protein” is theprotein product of a fusion gene.

[0059] “Inhibit” means to partially or completely block a particularprocess or activity. For example, a compound inhibits skeletal muscleatrophy if it either completely or partially prevents muscle atrophy.

[0060] As used herein, two DNA sequences are said to be “operablyassociated” if the nature of the linkage between the two DNA sequencesdoes not (1) result in the introduction of a frame-shift mutation, (2)interfere with the ability of a promoter region to direct thetranscription of the coding sequences, or (3) interfere with the abilityof the corresponding RNA transcript to be translated into a protein. Forexample, a coding sequence and regulatory sequences are operablyassociated when they are covalently linked in such a way as to place thetranscription of the coding sequence under the influence or control ofthe regulatory sequences. Thus, a promoter region is operably associatedwith a coding sequence when the promoter region is capable of effectingtranscription of that DNA sequence such that the resulting transcript iscapable of being translated into the desired protein or polypeptide.

[0061] “Percent identity” means the percentage of nucleotides or aminoacids that two sequences have in common, calculated as follows. Tocalculate the percent identity for a specific sequence (the query), therelevant part of the query sequence is compared to a reference sequenceusing the BestFit comparison computer program, Wisconsin Package,Version 10.1, available from the Genetics Computer Group, Inc. Thisprogram uses the algorithm of Smith and Waterman, Advances in AppliedMathematics, Issue 2: 482-489 (1981). Percent identity is calculatedwith the following default parameters for the BestFit program: thescoring matrix is blosum62.cmp, the gap creation penalty is 8 and thegap extension penalty is 2. When comparing a sequence to the referencesequence, the relevant part of the query sequence is that which isderived from a CRFR sequence. For example, where the query is aCRFR/purification tag fusion protein, only the CRFR polypeptide portionof the sequence is aligned to calculate the percent identity score.

[0062] “Polypeptide” means any chain of amino acids, regardless oflength or post-translational modification (e.g., phosphorylation orglycosylation).

[0063] “Promoter” means a DNA sequence which controls the initiation oftranscription and the rate of transcription from a gene or codingregion.

[0064] “Prophylactic treatment” means preventive treatment of a subject,not currently exhibiting signs of skeletal muscle atrophy, in order tocompletely or partially block the occurrence of skeletal muscle atrophy.One of skill in the art would recognize that certain individuals are atrisk for skeletal muscle atrophy as discussed in the background sectionherein. Furthermore, one of skill in the art would recognize that if thebiochemical changes leading to skeletal muscle atrophy are appropriatelyregulated, that the occurrence of atrophy would be prevented or reducedin at-risk individuals. For example, muscular dystrophy patientsbeginning treatment with corticosteroids are at risk for developingskeletal muscle atrophy indicating that prophylactic treatment of suchpatients would be appropriate.

[0065] “Regulate” in all its grammatical forms, means to increase,decrease or maintain, e.g., to regulate skeletal muscle mass or functionmeans to increase, decrease or maintain the level of skeletal musclemass or function.

[0066] “Regulation of skeletal muscle mass or function” includesregulation of skeletal muscle mass, skeletal muscle function or both.

[0067] “Regulatory element” means a DNA sequence that is capable ofcontrolling the level of transcription from an operably associated DNAsequence. Included within this definition of regulatory element arepromoters and enhancers. E.g., a CRFR gene regulatory element is a DNAsequence capable of controlling the level of transcription from the CRFRgene.

[0068] “Reporter gene” means a coding sequence whose product can bedetected, preferably quantitatively, wherein the reporter gene isoperably associated with a heterologous promoter or enhancer elementwhich is responsive to a signal which is to be measured. The promoter orenhancer element in this context is referred to herein as a “responsiveelement”.

[0069] “Selective agonist” means that the agonist has significantlygreater activity toward a certain receptor(s) compared with otherreceptors, not that it is completely inactive with regard to otherreceptors.

[0070] “Skeletal muscle hypertrophy” means an increase in skeletalmuscle mass or skeletal muscle function or both.

[0071] “Skeletal muscle atrophy” means the same as “muscle wasting” andmeans a decrease in skeletal muscle mass or skeletal muscle function orboth.

[0072] “Splice variant” means a mRNA or protein which results fromalternative exon usage. One of skill in the art recognizes that,depending on cell type, or even within a single cell type, a mRNA may beexpressed in a different form, as a splice variant, and thus thetranslated protein will be different depending upon the mRNA that isexpressed.

[0073] A “therapeutically effective amount” of a substance is an amountcapable of producing a medically desirable result in a treated patient,e.g., decreases skeletal muscle atrophy, increases skeletal muscle massor increases skeletal muscle function, with an acceptable benefit: riskratio; in a human or non-human mammal.

[0074] “Therapeutic treatment” means treatment of a subject in which anincrease in muscle mass or muscle function is desirable. For example,treatment of a subject currently exhibiting signs of skeletal muscleatrophy in order to partially or completely reverse the skeletal muscleatrophy that has occurred or to completely or partially block theoccurrence of further skeletal muscle atrophy would be therapeutictreatment of that subject. The term “therapeutic treatment” alsoincludes, for example, treatment of a subject not exhibiting signs ofskeletal muscle atrophy to induce skeletal muscle hypertrophy, e.g.,treatment of a livestock animal to increase muscle mass.

[0075] The term “treatment” means prophylactic or therapeutic treatment.

[0076] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the arts of protein chemistry, pharmacology, or molecularbiology. The methods, materials and examples described herein are notintended to be limiting. Other methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the present invention.

[0077] II. The Role of CRFRs in Regulation of Skeletal Muscle Mass

[0078] One of skill in the art would recognize the utility of thepresent invention given the information in the prior art and theteachings below. The results described herein demonstrate thatadministration of a CRF receptor agonist which activates both CRF₁R andCRF₂R (non-selective CRFR agonist) blocks and/or inhibits the skeletalmuscle atrophy inducing effect of denervation, disuse or dexamethasonetreatment in models of skeletal muscle atrophy. In addition, data showthat CRFR agonists do not show this anti-atrophy effect in mice in whichCRF₂R has been knocked out. Also, in rats in which the CRF₁R mediatedHPA axis has been interrupted by removal of the adrenal glands (surgicaladrenalectomy), treatment of these animals with the non-selective CRFRagonists shows an anti-atrophy effect, indicating that the CRF₂Rmediates the anti-atrophy effects. Furthermore, results demonstrate thatadministration of a non-selective CRFR agonist show a hypertrophyinducing effect. Together, these data demonstrate the modulatory role ofthe CRF₂R in the process of skeletal muscle atrophy. The specific roleof CRFRs in vivo was investigated using the pharmacological agents,sauvagine (Bachem Biosciences, Inc. King of Prussia, Pa.) and urocortin(Bachem Biosciences, Inc.), which are selective agonists for CRFRs invarious models of skeletal muscle atrophy, described hereinafter. Theseagents have been well characterized and are described in the scientificliterature.

[0079] FIGS. 1-7 and 9 show the results of experiments demonstratingthat administration of selective agonists of CRFRs results instatistically significant inhibition of skeletal muscle atrophy. FIG. 8shows that the anti-atrophy effect of the CRFR agonist, sauvagine, ismediated through CRF₂R. CRFR agonists administered twice daily incombination with the phosphodiesterase inhibitor, theophylline, resultedin inhibition of skeletal muscle atrophy in animal models of skeletalmuscle atrophy. Theophylline was added to potentiate the duration andmagnitude of action of the CRFR agonist therefore resulting in increasedefficacy of these compounds. Theophylline administered alone in theseatrophy models had no effect, demonstrating that the anti-atrophy effectof the CRFR agonist in combination with theophylline was due to theeffect of the CRFR agonist. Furthermore, continuous dosing of the CRFRagonist in the absence of theophylline, via osmotic mini-pump, alsoresulted in inhibition of skeletal muscle atrophy and/or in skeletalmuscle hypertrophy. Statistical significance of the results wasdetermined using ANCOVA (Douglas C. Montgomery, Design and Analysis ofExperiments, John Wiley and Sons, New York (2^(nd) ed. 1984)).Abbreviations used in FIGS. 1-9: g-gram; SEM-standard error of the mean.

[0080] Specifically, FIG. 1 (FIG. 1.) shows that sauvagine inhibitsdenervation-induced atrophy of the medial gastrocnemius muscle in amouse sciatic nerve denervation atrophy model. Legend: A—physiologicalsaline (control); B—sauvagine (0.01 mg/kg)+theophylline; C—sauvagine(0.03 mg/kg)+theophylline; D—sauvagine (0.1 mg/kg)+theophylline;E—sauvagine (1.0 mg/kg)+theophylline; *—p≦0.05 compared to saline.Following denervation of the right sciatic nerve, male mice wereinjected subcutaneously in the midscapular region twice daily withsauvagine, at the doses indicated above or vehicle control(physiological saline) for nine days. Sauvagine was co-administered with30 mg/kg theophylline. On day nine, the medial gastrocnemius muscle wasremoved and weighed to determine the degree of atrophy.

[0081]FIG. 2 (FIG. 2.) shows that sauvagine inhibits denervation-inducedatrophy of the tibialis anterior muscle in a mouse sciatic nervedenervation atrophy model. Legend: A—water (control); B—sauvagine (0.1mg/kg/d); C—sauvagine (0.3 mg/kg/d); D—sauvagine (1.0 mg/kg/d); *—p≦0.05compared to water. Following denervation of the right sciatic nerve,male mice were dosed with either sauvagine or vehicle control(physiological saline) by continuous infusion using an Alzet osmoticminipump at 5 μl/hr until the end of the experimental period (withoutadditional theophylline). The daily delivered dose of sauvagine isindicated above. Minipump implantation was performed at the time ofsciatic nerve denervation. On day nine the tibialis anterior muscle wasremoved and weighed to determine the degree of atrophy.

[0082]FIG. 3 (FIG. 3.) demonstrates that sauvagine inhibitsglucocorticoid-induced muscle atrophy of the tibialis anterior (FIG. 3A)and medial gastrocnemius muscles (FIG. 3B) in the mouseglucocorticoid-induced atrophy model. Legend: A—water only with nodexamethasone included in drinking water (non-atrophied control);B—water+dexamethasone (atrophied control); C—sauvagine (0.1mg/kg/d)+dexamethasone; D—sauvagine (0.3 mg/kg/d)+dexamethasone;E—sauvagine (1.0 mg/kg/d)+dexamethasone; *—p≦0.05 compared to water;#—p≦0.05 compared to water+dexamethasone. Following the addition of theglucocorticoid, dexamethasone, to the drinking water (1.2 mg/kg/d), malemice were dosed with the above indicated agents or vehicle control(physiological saline) by continuous infusion using an Alzet osmoticminipump at 5 μl/hr until the end of the experimental period (withoutadditional theophylline). The daily delivered dose of sauvagine is asindicated above. Minipump implantation was performed at the time ofinitiation of dexamethasone exposure. Nine days following the initiationof dosing sauvagine, the medial gastrocnemius and tibialis anteriormuscles were removed and weighed to determine the degree of atrophy.

[0083]FIG. 4 (FIG. 4.) demonstrates that sauvagine inhibitsdisuse-induced atrophy of the tibialis anterior (FIG. 4A) and medialgastrocnemius (FIG. 4B) muscles. In addition, statistically significanthypertrophy of the medial gastrocnemius and tibialis anterior muscles ofthe non-casted leg was also observed with sauvagine treatment. Legend:A—physiological saline (control); B—theophylline; C—sauvagine (0.03mg/kg)+theophylline; D—sauvagine (0.1 mg/kg)+theophylline; E—sauvagine(0.3 mg/kg)+theophylline; *—p≦0.05 compared to saline. Following castingof the right hind leg, male mice were injected subcutaneously in themidscapular region twice daily, with sauvagine or vehicle control(physiological saline) for ten days at the daily delivered doseindicated. Sauvagine was co-administered with twice dailyintra-peritoneal dosing of the phosphodiesterase inhibitor theophylline(30 mg/kg). On day ten, the medial gastrocnemius and tibialis anteriormuscles were removed and weighed to determine the degree of atrophy.

[0084]FIG. 5 (FIG. 5.) demonstrates that both sauvagine and urocortininhibit denervation-induced atrophy of the tibialis anterior muscle, ina mouse sciatic nerve denervation atrophy model. In addition,hypertrophy of the non-denervated leg was observed with urocortintreatment. Legend: A—water (control); B—sauvagine (1 mg/kg/d);C—urocortin (1.0 mg/kg/d); *—p≦0.05 compared to water. Followingdenervation of the right sciatic nerve, male mice were dosed with theabove indicated agents or vehicle control (physiological saline) bycontinuous infusion using an Alzet osmotic minipump at 5 μl/hr until theend of the experimental period (without additional theophylline). Thedaily delivered dose of the agents is indicated above. Minipumpimplantation was performed at the same time as the sciatic nervedenervation. On day nine the tibialis anterior muscle was removed andweighed to determine the degree of atrophy.

[0085]FIG. 6 (FIG. 6.) demonstrates that urocortin inhibitsdisuse-induced atrophy of the tibialis anterior (FIG. 6A) and medialgastrocnemius (FIG. 6B) muscles in the mouse leg casting disuse atrophymodel. Legend: A—physiological saline (control); B—urocortin (0.3mg/kg)+theophylline; *—p≦0.05 compared to saline. Following casting ofthe right hind leg, male mice were injected subcutaneously in themidscapular region twice daily, with urocortin or vehicle control(physiological saline) for ten days. Urocortin was administered at thedoses indicated in the description of FIGS. 6A and 6B. Urocortin wasco-administered with twice daily intra-peritoneal dosing of thephosphodiesterase inhibitor theophylline (30 mg/kg). On day ten, themedial gastrocnemius and tibialis anterior muscles were removed andweighed to determine the degree of atrophy.

[0086]FIG. 7 (FIG. 7) demonstrates that sauvagine inhibitsdenervation-induced atrophy of the tibialis anterior (FIG. 7A), EDL(FIG. 7B), soleus (FIG. 7C), medial gastrocnemius (FIG. 7D), andplantaris (FIG. 7E) muscles. In addition, sauvagine caused statisticallysignificant hypertrophy of the non-denervated EDL muscle (FIG. 7B).Legend: A—physiological saline (control); B—sauvagine (0.003mg/kg)+theophylline; C—sauvagine (0.01 mg/kg)+theophylline; D—sauvagine(0.03 mg/kg)+theophylline; #—p≦0.05 compared to corresponding controls.Following denervation of the right sciatic nerve, male adrenalectomizedrats (adrenalectomized rats were used to remove the skeletal muscleatrophy-inducing effects of activation of the HPA axis via agonisms ofthe CRF₁R) were injected subcutaneously in the midscapular region twicedaily, with either sauvagine or vehicle control (physiological saline)for nine days at the doses shown above. Sauvagine was co-administeredwith 30 mg/kg theophylline. On day nine, the tibialis anterior, extensordigitorum longus (EDL), soleus, medial gastrocnemius, and plantarismuscles were removed and weighed to determine the degree of atrophy.

[0087]FIG. 8 (FIG. 8.) demonstrates that sauvagine inhibits the atrophyobserved in wild-type but not CRF₂R knockout mice in the mouse sciaticnerve denervation atrophy model. Legend: A-C—wild-type mice; D-F—CRF₂Rknockout mice. A and D—water (control); B and E—sauvagine (0.3 mg/kg/d);C and F—sauvagine (1.0 mg/kg/d); *—p≦0.05 compared to saline. Followingdenervation of the right sciatic nerve, female wild-type and CRF₂Rknockout mice were dosed with sauvagine or vehicle control by continuousinfusion using an Alzet osmotic minipump at 5 μl/hr for nine days at thedaily delivered dose indicated above. On day nine, the tibialis anteriormuscle was removed and weighed to determine the degree of atrophy.

[0088]FIG. 9 (FIG. 9) demonstrates that sauvagine inhibitsdisuse-induced loss of EDL and soleus muscle mass (FIG. 9A) and inhibitsloss of muscle function as assessed by measurement of absolute force(FIG. 9B) in the mouse leg casting disuse atrophy model. Legend: A—noncasted muscle control; B—casted muscle, saline control; C—casted muscle,sauvagine (0.3 mg/kg)+theophylline (30 mg/kg); *—p≦0.05 compared tosaline. Following casting of the right hind leg, male mice were injectedsubcutaneously in the midscapular region twice daily, with eithersauvagine or vehicle control (physiological saline) for ten days at thedoses indicated above. Sauvagine was co-administered 30 mg/kgtheophylline. On day ten, the EDL and soleus muscles were removed andabsolute force and mass measurements taken to determine the degree ofatrophy.

[0089] III. Preparation of CRFRs, CRF or CRF Analogs, or Cell LinesExpressing CRFRs

[0090] CRF₁R, CRF₂R, CRF and CRF analogs can be prepared for a varietyof uses, including, but not limited to, the generation of antibodies,use as reagents in the screening assays of the present invention, anduse as pharmaceutical reagents for the treatment of skeletal muscleatrophy. It will be clear to one of skill in the art that, for certainembodiments of the invention, purified polypeptides will be most useful,while for other embodiments cell lines expressing the polypeptides willbe most useful. For example, in situations where it is important toretain the structural and functional characteristics of the CRFR, e.g.,in a screening method to identify candidate compounds which activateCRFRs, it is desirable to use cells which express functional CRFRs.

[0091] Because CRF and CRF analogs are short polypeptides, the skilledartisan will recognize that these polypeptides will be most convenientlyprovided by direct synthesis, rather than by recombinant means, usingtechniques well known in the art. In addition, many of these moleculesare commercially available.

[0092] Where the source of CRFRs is a cell line expressing thepolypeptide, the cells may, for example, endogenously express CRFR, havebeen stimulated to increase endogenous CRFR expression or have beengenetically engineered to express a CRFR. Methods for determiningwhether a cell line expresses a polypeptide of interest are known in theart, for example, detection of the polypeptide with an appropriateantibody, use of a DNA probe to detect mRNA encoding the protein (e.g.,northern blot or PCR techniques), or measuring binding of an agentselective for the polypeptide of interest (e.g., a radiolabeledselective agonist).

[0093] The use of recombinant DNA technology in the preparation ofCRF₁R, CRF₂R, or of cell lines expressing these polypeptides isparticularly contemplated. Such recombinant methods are well known inthe art. To express recombinant CRF₁R or CRF₂R, an expression vectorthat comprises a nucleic acid which encodes the polypeptide of interestunder the control of one or more regulatory elements, is prepared.Genomic or cDNA sequences encoding CRF₁R and CRF₂R from several specieshave been described and are readily available from the GenBank database(available at <http://www.ncbi.nlm.nih.gov/>) or Derwent database(available at <http://www.derwent.co.uk/geneseq/lndex.html>) as well asin the sequence listing for this application. The accession numbers forCRF₁R and CRF₂R sequences and corresponding SEQ ID NOS. are shown inTable I. Using this publicly available sequence information, one meansof isolating a nucleic acid molecule encoding a CRF₁R or CRF₂R is toscreen a genomic DNA or cDNA library with a natural or artificiallysynthesized DNA probe, using methods well known in the art, e.g., by PCRamplification of the sequence from an appropriate library. Anothermethod is to use oligonucleotide primers specific for the receptor ofinterest to PCR amplify the cDNA directly from mRNA isolated from aparticular tissue (such as skeletal muscle). Such isolated mRNA iscommercially available. One of skill in the art would also recognizethat by using nucleic acid probes corresponding to portions of the knownCRFR receptor sequences the homologous cDNAs or genomic sequences fromother species can be obtained using known methods. Particularly usefulin the methods of the present invention are CRFR receptors from thespecies including, but not limited to, human, mouse, rat, pig, monkey,chimpanzee, marmoset, dog, cow, sheep, cat, chicken and turkey. Bymethods well known in the art, the isolated nucleic acid moleculeencoding the CRFR of interest is then ligated into a suitable expressionvector. The expression vector, thus prepared, is expressed in a hostcell and the host cells expressing the receptor are used directly in ascreening assay or the receptor is isolated from the host cellsexpressing the receptor and the isolated receptor is used in a screeningassay.

[0094] The host-expression vector systems that may be used for purposesof the invention include, but are not limited to: microorganisms such asbacteria (e.g., E. coli, B. subtilis) transformed with recombinantbacteriophage DNA, plasmid DNA, or cosmid DNA expression vectorscontaining CRFR nucleotide sequences; yeast (e.g., Saccharomyces,Pichia) transformed with recombinant yeast expression vectors containingCRFR nucleotide sequences; insect cell systems infected with recombinantvirus expression vectors (e.g., baculovirus) containing CRFR nucleotidesequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, tobacco mosaic virus) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing CRFR nucleotide sequences; or mammalian cell systems(e.g., COS, CHO, HEK293, NIH3T3) harboring recombinant expressionconstructs containing promoters derived from the genome of mammaliancells (e.g., metallothionein promoter) or from mammalian viruses (e.g.,retrovirus LTR) and also containing CRFR nucleotide sequences.

[0095] The host cell is used to produce the polypeptide of interest.Because the CRFR is a membrane bound molecule, it is purified from thehost cell membranes or the CRFR is utilized while anchored in the cellmembrane, i.e., whole cells or membrane fractions of cells are used.Purification or enrichment of the CRFRs from such expression systems isaccomplished using appropriate detergents and lipid micelles by methodswell known to those skilled in the art.

[0096] In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the geneproduct being expressed. For example, when a large quantity of suchprotein is produced for the generation of antibodies to CRFRs, vectorswhich direct the expression of high levels of protein products aredesirable. One skilled in the art is able to generate such vectorconstructs and purify the proteins by a variety of methodologiesincluding selective purification technologies such as fusion proteinselective columns and antibody columns, and non-selective purificationtechnologies.

[0097] In an insect protein expression system, the baculovirus A.californica nuclear polyhedrosis virus (AcNPV), is used as a vector toexpress foreign genes in S. frugiperda cells. In this case, CRFRnucleotide sequences are cloned into non-essential regions of the virusand placed under the control of an AcNPV promoter. The recombinantviruses are then used to infect cells in which the inserted gene isexpressed and the protein is purified by one of many techniques known toone skilled in the art.

[0098] In mammalian host cells, a number of viral-based expressionsystems may be utilized. Utilization of these expression systems oftenrequires the creation of specific initiation signals in the vectors forefficient translation of the inserted nucleotide sequences. This isparticularly important if a portion of the CRFR gene is used which doesnot contain the endogenous initiation signal. The placement of thisinitiation signal, in frame with the coding region of the insertednucleotide sequence, as well as the addition of transcription andtranslation enhancing elements and the purification of the recombinantprotein, are achieved by one of many methodologies known to one skilledin the art. Also important in mammalian host cells is the selection ofan appropriate cell type which is capable of the necessary posttranslational modifications of the recombinant protein. Suchmodifications, for example, cleavage, phosphorylation, glycosylation,etc., require the selection of the appropriate host cell which containsthe modifying enzymes. Such host cells include, but are not limited to,CHO, HEK293, NIH3T3, COS, etc. and are known by those skilled in theart.

[0099] For long term, high expression of recombinant proteins, stableexpression is preferred. For example, cell lines that stably expressCRFRs may be engineered. One of skill in the art, following knownmethods such as electroporation, calcium phosphate transfection, orliposome-mediated transfection, can generate a cell line that stablyexpresses CRFRs. This is usually accomplished by transfecting cellsusing expression vectors which contain appropriate expression controlelements (e.g., promoter sequences, enhancer sequences, transcriptionaltermination sequences, polyadenylation sites, translational start sites,etc.), a selectable marker, and the gene of interest. The selectablemarker may either be contained within the same vector, as the gene ofinterest, or on a separate vector, which is co-transfected with the CRFRsequence containing vector. The selectable marker in the expressionvector may confer resistance to the selection and allows cells to stablyintegrate the vector into their chromosomes and to grow to form fociwhich in turn can be cloned and expanded into cell lines. Alternatively,the expression vector may allow selection of the cell expressing theselectable marker utilizing a physical attribute of the marker, i.e.,expression of Green Fluorescent Protein (GFP) allows for selection ofcells expressing the marker using fluorescence activated cell sorting(FACS) analysis.

[0100] One of skill in the art is able to select an appropriate celltype for transfection in order to allow for selection of cells intowhich the gene of interest has been successfully integrated. Forexample, where the selectable marker is herpes simplex virus thymidinekinase, hypoxanthine-guanine phosphoribosyltransferase or adeninephosphoribosyltransferase, the appropriate cell type would be tk-,hgprt- or aprt-cells, respectively. Or, normal cells can be used wherethe selectable marker is dhfr, gpt, neo or hygro which confer resistanceto methotrexate, mycophenolic acid, G-418 or hygromycin, respectively.Such recombinant cell lines are useful for identification of candidatecompounds that affect the CRFR activity.

[0101] IV. Preparation of CRFR Antibodies

[0102] Antibodies that selectively recognize one or more epitopes of aCRFR are also encompassed by the invention. Such antibodies include,e.g., polyclonal antibodies, monoclonal antibodies, chimeric antibodies,human antibodies, single chain antibodies, Fab fragments, F(ab′)₂fragments, molecules produced using a Fab expression library, humanantibodies (polyclonal or monoclonal) produced in transgenic mice andepitope binding fragments of any of the above. For therapeutic uses,chimeric or human antibodies are preferred; human antibodies are mostpreferred.

[0103] The antibodies can be utilized in conjunction with the compoundscreening schemes described herein for the evaluation of test compounds,e.g., for immobilization of CRFR polypeptides or such antibodies can beused in conjunction with gene therapy techniques to evaluate, forexample, the expression of CRFRs either in cells or directly in patienttissues in which these genes have been introduced. In addition,antibodies of the present invention are useful in the treatment ofskeletal muscle atrophy. Antibodies selective for the CRFR can bescreened by the methods of the present invention to identify a subset ofthe antibodies that are CRFR agonists. In addition, anti-idiotypeantibodies generated against antibodies specific for CRF or a CRF analogmay be useful as CRFR agonists and like anti-CRFR antibodies may bescreened for their ability to activate the CRFR by methods of thepresent invention.

[0104] For the production of antibodies, a variety of host animals maybe immunized by injection with CRFR, CRF or a CRF analog, anti-CRFantibody, anti-CRF analog antibody, or immunogenic fragments thereof bymethods well known in the art. For preparation of an anti-idiotypeantibody the immunogen is an anti-CRF antibody or anti-CRF analogantibody. Production of anti-idiotype antibodies is described, forexample, in U.S. Pat. No. 4,699,880, incorporated herein by reference.Suitable host animals include, but are not limited to, rabbits, mice,goats, sheep and horses. Immunization techniques are well known in theart. Polyclonal antibodies can be purified from the serum of theimmunized animals, or monoclonal antibodies can be generated by methodsthat are well known in the art. These techniques include, but are notlimited to, the well-known hybridoma techniques of Kohler and Milstein,human B-cell hybridoma techniques, and the EBV hybridoma technology.Monoclonal antibodies may be of any immunoglobulin class, including IgG,IgE, IgM, IgA, and IgD containing either kappa or lambda light chains.

[0105] Because of the immunogenicity of non-human antibodies in humans,chimeric antibodies are preferred to non-human antibodies when used fortherapeutic treatment of human patients. Techniques of producing andusing chimeric antibodies are known in the art, and are described in,for example, U.S. Pat. Nos. 5,807,715; 4,816,397; 4,816,567; 5,530,101;5,585,089; 5,693,761; 5,693,762; 6,180,370; and 5,824,307, allincorporated herein by reference.

[0106] Completely human antibodies are particularly desirable fortherapeutic treatment of human patients because they are lessimmunogenic than non-human antibodies or chimeric antibodies. Suchantibodies can be produced using transgenic mice which are substantiallyincapable of expressing endogenous immunoglobulin heavy and light chaingenes, but which can express human heavy and light chain genes. Thetransgenic mice are immunized in the normal fashion with a selectedantigen, e.g., all or a portion of CRF₂R. Monoclonal antibodies directedagainst the antigen are obtained using conventional hybridoma technologyfrom these immunized transgenic mice. This technology is described indetail in U.S. Pat. Nos. 5,874,299; 5,877,397; 5,569,825; 5,661,016;5,770,429; and 6,075,181, all incorporated herein by reference. As analternative to obtaining human immunoglobulins directly from the cultureof the hybridoma cells, the hybridoma cells can be used as a source ofrearranged heavy chain and light chain loci for subsequent expression orgenetic manipulation. Isolation of genes from such antibody-producingcells is straightforward since high levels of the appropriate mRNAs areavailable. The recovered rearranged loci can be manipulated as desired.For example, the constant region can be eliminated or exchanged for thatof a different isotype or the variable regions can be linked to encodesingle chain Fv regions. Such techniques are described in WO 96/33735and WO 96/34096, all incorporated herein by reference.

[0107] V. Selection of Test Compounds

[0108] Compounds that can be screened in accordance with the assays ofthe invention include but are not limited to, libraries of knowncompounds, including natural products, such as plant or animal extracts,synthetic chemicals, biologically active materials including proteins,peptides such as soluble peptides, including but not limited to membersof random peptide libraries and combinatorial chemistry derivedmolecular library made of D- or L-configuration amino acids,phosphopeptides (including, but not limited to, members of random orpartially degenerate, directed phosphopeptide libraries), antibodies(including, but not limited to, polyclonal, monoclonal, chimeric, human,anti-idiotypic or single chain antibodies, and Fab, F(ab′)₂ and Fabexpression library fragments, and epitope-binding fragments thereof),organic and inorganic molecules.

[0109] In addition to the more traditional sources of test compounds,computer modeling and searching technologies permit the rationalselection of test compounds by utilizing structural information from theligand binding site of CRFR or from already identified agonists ofCRFRs. Such rational selection of test compounds can decrease the numberof test compounds that must be screened in order to identify a candidatetherapeutic compound. CRFRs are GPCRs, and thus knowledge of the CRFRprotein sequence allows for the generation of a model of its bindingsite that can be used to screen for potential ligands. This process canbe accomplished in several manners well known in the art. Briefly, themost robust approach involves generating a sequence alignment of theCRFR sequence to a template (derived from the bacterio-rhodopsin orrhodopsin crystal structures or other GPCR model), conversion of theamino acid structures and refining the model by molecular mechanics andvisual examination. If a strong sequence alignment cannot be obtainedthen a model may also be generated by building models of the hydrophobichelices. These are then fitted together by rotating and translating eachhelix relative to the others starting from the general layout of theknown rhodopsin structures. Mutational data that point towardsresidue-residue contacts may also be used to position the helicesrelative to each other so that these contacts are achieved. During thisprocess, docking of the known ligands into the binding site cavitywithin the helices may also be used to help position the helices bydeveloping interactions that would stabilize the binding of the ligand.The model may be completed by refinement using molecular mechanics andloop building of the intracellular and extracellular loops usingstandard homology modeling techniques. General information regardingGPCR structure and modeling can be found in Schoneberg, T. et. al.,Molecular and Cellular Endocrinology, 151:181-193 (1999), Flower, D.,Biochimica et Biophysica Acta, 1422:207-234 (1999), and Sexton, P. M.,Current Opinion in Drug Discovery and Development, 2(5):440-448 (1999).

[0110] Once the model is completed, it can be used in conjunction withone of several existing computer programs to narrow the number ofcompounds to be screened by the screening methods of the presentinvention. The most general of these is the DOCK program (UCSF MolecularDesign Institute, 533 Parnassus Ave, U-64, Box 0446, San Francisco,Calif. 94143-0446). In several of its variants it can screen databasesof commercial and/or proprietary compounds for steric fit and roughelectrostatic complementarity to the binding site. It has frequentlybeen found that molecules that score well within DOCK have a betterchance of being ligands. Another program that can be used is FLEXX(Tripos Inc., 1699 South Hanley Rd., St. Louis, Mo., 63144-2913(www.tripos.com)). This program, being significantly slower, is usuallyrestricted to searches through smaller databases of compounds. Thescoring scheme within FLEXX is more detailed and usually gives a betterestimate of binding ability than does DOCK. FLEXX is best used toconfirm DOCK suggestions, or to examine libraries of compounds that aregenerated combinatorially from known ligands or templates.

[0111] VI. Screening Assays to Identify Candidate Compounds for theRegulation of Skeletal Muscle Mass or Function

[0112] The finding that CRF₂R plays a role in regulating skeletal muscleatrophy enables various methods of screening one or more test compoundsto identify candidate compounds that ultimately may be used forprophylactic or therapeutic treatment of skeletal muscle atrophy. Thisinvention provides methods for screening test compounds for theirability to bind to CRF₂R, activate CRF₂R, prolong or augment theagonist-induced activation of CRF₂R or of a CRF₂R signal transductionpathway or increase expression of CRF₂R or CRF genes.

[0113] Because CRF₂R and CRF₁R are homologous proteins, it is expectedthat a certain proportion of agonists for CRF₂R will also function asagonists of CRF₁R. As discussed above, activation of CRF₁R inducesactivation of the HPA axis and concomitant production ofcorticosteroids. In most cases in which an increase in muscle mass orfunction is desired, it is not desirable to activate the HPA axis.Therefore, in addition to screening test compounds for their ability toactivate CRF₂R, the invention also provides for the use of CRF₂R andCRF₁R to screen for selective agonists of CRF₂R. When selectingcandidate compound useful for the treatment of acute or chronic muscleatrophy, which is not related to muscular dystrophy, it is preferablethat the candidate compounds be selective for CRF₂R. Preferably thecandidate compound exhibits 10-fold selectivity for CRF₂R versus CRF₁R(i.e., 10-fold more active against CRF₂R than against CRF₁R), morepreferably 100-fold selectivity and most preferably 1000-fold or greaterselectivity. As published studies have demonstrated a benefit ofcorticosteroid therapy in the treatment of muscular dystrophies, it maybe beneficial that a CRF₂R agonist retain some level of CRF₁R agonismwhen used to treat muscular dystrophies. Thus, for the treatment ofmuscular dystrophies, a compound of lower selectivity that activates theCRF₂R as well as the CRF₁R, over a similar concentration range, ispreferred. Preferably the candidate compound is 100-fold selective forCRF₂R versus CRF₁R, more preferably 10-fold selective and mostpreferably not selective for CRF₂R versus CRF₁R (i.e., the activity ofthe candidate compound is substantially similar for CRF₂R and CRF₁R).Also, in this case, it may be more preferable that the compound is fullagonist for CRF₂R while being a partial agonist for CRF₁R. Such acandidate compound would therefore have a built-in limit to the maximumdegree of cortisol elevation and potential for muscle atrophy, while theanti-atrophy effect mediated through the CRF₂R could be enhanced byincreasing the dose. One of skill in the art would be able to readilydetermine whether a candidate compound is a full or partial agonist ofthe CRF₁R or CRF₂R using methods known in the art.

[0114] For screening for compounds which ultimately will be used toregulate skeletal muscle mass or function through CRF₂R in humans, it ispreferred that the initial in vitro screen be carried out using a CRF₂Rwith an amino acid sequence that is greater than 80% identical to SEQ IDNO:10 and more preferably greater than 90% identical to SEQ ID NO:10.More preferably the test compounds will be screened against a human,mouse or rat CRF₂R, with the most preferable being human. For screeningfor compounds which ultimately will be used to regulate skeletal musclemass or function through CRF₂R in a non-human species it is preferableto use the CRF₂R from the species in which treatment is contemplated.

[0115] For screening to determine the level of activity that a test orcandidate compound has toward CRF₁R to determine what, if any,selectivity a candidate compound exhibits for CRF₂R versus CRF₁R, it ispreferred that the initial screen be carried out using a CRF₁R with anamino acid sequence that is greater than 80% identical to SEQ ID NO:2and more preferably greater than 90% identical to SEQ ID NO:2. Morepreferably the test compounds will be screened against a human, mouse orrat CRF₁R, with the most preferable being human. For screening forcompounds which ultimately will be used to regulate skeletal muscle massor function in a non-human species, it is preferable to use the CRF₁Rfrom the species in which treatment is contemplated.

[0116] The methods of the present invention are amenable to highthroughput applications; however, the use of as few as one test compoundin the method is encompassed by the term “screening”. Test compoundswhich bind to CRF₂R, activate CRF₂R, prolong or augment theagonist-induced activation of CRF₂R or of a CRF₂R signal transductionpathway, or increase expression of CRF₂R or CRF genes, as determined bya method of the present invention, are referred to herein as “candidatecompounds.” Such candidate compounds can be used to regulate skeletalmuscle mass or function. However, more typically, this first level of invitro screen provides a means by which to select a narrower range ofcompounds, i.e., the candidate compounds, which merit furtherinvestigation in additional levels of screening. The skilled artisanwill recognize that a utility of the present invention is to identify,from a group of one or more test compounds, a subset of compounds whichmerit further investigation. One of skill in the art will also recognizethat the assays of the present invention are useful in ranking theprobable usefulness of a particular candidate compound relative to othercandidate compounds. For instance, a candidate compound which activatesCRF₂R at 1000 nM (but not at 10 nM) is of less interest than one whichactivates CRF₂R at 10 nM. Using such information the skilled artisan mayselect a subset of the candidate compounds, identified in the firstlevel of screening, for further investigation. By the way of exampleonly, compounds which activate CRF₂R at concentrations of less than 200nM might be further tested in an animal model of skeletal muscleatrophy, whereas those above that threshold would not be further tested.The skilled artisan will also recognize that, depending on how the groupof test compounds is selected, and how the positives are selected, onlya certain proportion of test compounds will be identified as candidatecompounds, and that this proportion may be very small.

[0117] The assay systems described below may be formulated into kitscomprising CRF₂R or cells expressing the CRF₂R which can be packaged ina variety of containers, e.g., vials, tubes microtitre well plates,bottles and the like. Other reagents can be included in separatecontainers and provided with the kit, e.g., positive control samples,negative control samples, buffers and cell culture media.

[0118] In one embodiment, the invention provides a method for screeningone or more test compounds to identify candidate compounds that bind toCRF₂R. Methods of determining binding of a compound to a receptor arewell known in the art. Typically, the assays include the steps ofincubating a source of the CRF₂R with a labeled compound, known to bindto the receptor, in the presence or absence of a test compound anddetermining the amount of bound labeled compound. The source of CRF₂Rmay either be cells expressing CRF₂R or some form of isolated CRF₂R, asdescribed herein. The labeled compound can be CRF or any CRF analoglabeled such that it can be measured, preferably quantitatively (e.g.,¹²⁵I-labeled, europium labeled, fluorescein labeled, GFP labeled,³⁵S-methionine labeled). Such methods of labeling are well known in theart. Test compounds that bind to the CRFR cause a reduction in theamount of labeled ligand bound to the receptor, thereby reducing thesignal level compared to that from control samples (absence of testcompound). Variations of this technique have been described in whichreceptor binding in the presence and absence of G-protein uncouplingagents can discriminate agonists from antagonists (e.g., binding in theabsence and presence of a guanine nucleotide analog i.e., GpppNHp). SeeKeen, M., Radioligand Binding Methods for Membrane Preparations andIntact cells in Receptor Signal Transduction Protocols, R. A. J.Challis, (ed), Humana Press Inc., Totoway N.J. (1997).

[0119] Because it is desirable to discriminate between compounds whichbind specifically to CRF₂R, as compared with CRF₁R, the assays describedabove should be conducted using a cell, or membrane from a cell, whichexpresses only CRF₂R or the assays can be conducted with a recombinantsource of CRF₂R. Cells expressing both forms of CRFR may be modifiedusing homologous recombination to inactivate or otherwise disable theCRF₁R gene. Alternatively, if the source of CRFR contains more than oneCRFR type, the background signal produced by the receptor which is notof interest must be subtracted from the signal obtained in the assay.The background response can be determined by a number of methods,including elimination of the signal from the CRFR which is not ofinterest by use of antisense, antibodies or selective antagonists. Knownantagonists of CRFRs include antalarmin (CRF₁R selective),antisauvagine-30 (CRF₂R selective) and astressin (nonselective forCRF₁R/CRF₂R).

[0120] In another embodiment, the invention provides methods forscreening test compounds to identify candidate compounds which activateCRF₂R and/or CRF₁R. Typically, the assays are cell-based; however,cell-free assays are known which are able to differentiate agonist andantagonist binding as described above. Cell-based assays include thesteps of contacting cells which express CRF₁R or CRF₂R with a testcompound or control and measuring activation of the CRFR by measuringthe expression or activity of components of the CRFR signal transductionpathways.

[0121] As described in the background section above, CRFRs appear tocouple through several different pathways including G_(αs), G_(αq) orG_(αi), depending upon the cell type. It is thought that agonistactivation of CRFR allows the receptor to signal via any of thesepathways, provided that the necessary pathway components are present inthe particular cell type. Thus, to screen for CRFR activation, an assaycan use any of the signal transduction pathways as the readout even ifthe relevant cell type for treatment, in vivo, couples CRFR to skeletalmuscle atrophy via a different pathway. One of ordinary skill in the artwould recognize that a screening assay would be effective foridentifying useful CRFR agonists independent of the pathway by whichreceptor activation was measured. Assays for measuring activation ofthese signaling pathways are known in the art.

[0122] For example, after contact with the test compound, lysates of thecells can be prepared and assayed for induction of cAMP. cAMP is inducedin response to G_(αs) activation. Because G_(αs) is activated byreceptors other than CRFR and because a test compound may be exertingits effect through CRFRs or by another mechanism, two controlcomparisons are relevant for determining whether a text compoundincreases levels of cAMP via activation of a CRFR. One control comparesthe cAMP level of cells contacted with a test compound and the cAMPlevel of cells contacted with a control compound (i.e., the vehicle inwhich the test compound is dissolved). If the test compound increasescAMP levels relative to the control compound this indicates that thetest compound is increasing cAMP by some mechanism. The other controlcompares the cAMP levels of a CRFR expressing cell line and a cell linethat is essentially the same except that it does not express the CRFR,where both of the cell lines have been treated with test compound. Ifthe test compound elevates cAMP levels in the CRFR expressing cell linerelative to the cell line that does not express CRFRs, this is anindication that the test compound elevates cAMP via activation of theCRFRs.

[0123] In a specific embodiment of the invention, cAMP induction ismeasured with the use of DNA constructs containing the cAMP responsiveelement linked to any of a variety of reporter genes can be introducedinto cells expressing CRFRs. Such reporter genes include, but are notlimited to, chloramphenicol acetyltransferase (CAT), luciferase,glucuronide synthetase, growth hormone, fluorescent proteins (e.g.,Green Fluorescent Protein), or alkaline phosphatase. Following exposureof the cells to the test compound, the level of reporter gene expressioncan be quantitated to determine the test compound's ability to increasecAMP levels and thus determine a test compounds ability to activate theCRFR.

[0124] The cells useful in this assay are the same as for the CRFRbinding assay described above, except that cells utilized in theactivation assays preferably express a functional receptor which gives astatistically significant response to CRF or one or more CRF analog. Inaddition to using cells expressing full length CRFRs, cells can beengineered which express CRFRs containing the ligand binding domain ofthe receptor coupled to, or physically modified to contain, reporterelements or to interact with signaling proteins. For example, awild-type CRFR or CRFR fragment can be fused to a G-protein resulting inactivation of the fused G-protein upon agonist binding to the CRFRportion of the fusion protein. (Siefert, R. et al., Trends Pharmacol.Sci. 20: 383-389 (1999)). The cells should also preferably possess anumber of characteristics, depending on the readout, to maximize theinductive response by CRF or the CRF analog, for example, for detectinga strong induction of a CRE reporter gene; (a) a low natural level ofcAMP; (b) G proteins capable of interacting with CRFRs; (c) a high levelof adenylyl cyclase; (d) a high level of protein kinase A; (e) a lowlevel of phosphodiesterases; and (f) a high level of cAMP responseelement binding protein would be advantageous. To increase the responseto CRF or a CRF analog, host cells could be engineered to express agreater amount of favorable factors or a lesser amount of unfavorablefactors. In addition, alternative pathways for induction of the CREreporter could be eliminated to reduce basal levels.

[0125] In some instances, G protein-coupled receptor responses subside,or become desensitized, after prolonged exposure to an agonist. Anotherembodiment of the invention provides methods for identifying compoundsthat prolong or augment the agonist-induced activation of CRF₂R, or theCRF₂R signal transduction pathway, in response to a CRF₂R agonist. Suchcompounds may be used, for example, in conjunction with a CRF₂R agonistfor the treatment of skeletal muscle atrophy. Typically the method usesa cell based assay comprising in any order or concurrently (i)contacting the cells with a test compound; (ii) treating cellsexpressing functional CRF₂R with a CRF₂R agonist at a concentration ofagonist and for a period of agonist-receptor exposure sufficient toallow desensitization of the receptor; followed by (iii) determining thelevel of activation of the CRF₂R. One of skill in the art will recognizethat several mechanisms contribute to receptor desensitizationincluding, but not limited to, receptor phosphorylation, receptorinternalization or degradation and CRFR signal transduction pathwaydown-modulation. One of skill in the art can determine the appropriatetime (i.e., before, during or after agonist treatment) for contactingthe cells with the test compounds depending upon which mechanism ofdesensitization is targeted. For example, contacting the cells with testcompounds following agonist treatment, can detect test compounds whichblock receptor desensitization which occurs as a result ofphosphorylation of the receptor.

[0126] In another embodiment, the invention provides a method ofscreening one or more test compound to identify candidate compoundswhich regulate transcription from the CRF₂R gene or regulate CRF₂Rexpression. Candidate compounds which regulate transcriptional activityof CRFR genes may be identified using a reporter gene operablyassociated with a CRF₂R regulatory region (reporter gene construct).Such methods are known in the art. In one such method, the reporter geneconstruct is contacted with a test compound in the presence of a sourceof cellular factors and the level of reporter gene expression isdetermined. A test compound which causes an increase in the level ofexpression, compared to a control sample, is indicative of a candidatecompound which increases transcription of the CRF₂R gene. To provide thecellular factors required for in vitro or in vivo transcription,appropriate cells or cell extracts are prepared from any cell type thatnormally expresses CRF₂R.

[0127] Candidate compounds which regulate CRF₂R expression can also beidentified in a method wherein a cell is contacted with a test compoundand the expression of CRFR is determined. The level of expression ofCRF₂R in the presence of the test compound is compared with the level ofexpression in the absence of the test compound. Test compounds whichincrease the expression of CRF₂R are identified as candidate compoundsfor increasing muscle mass or muscle function. Such a method detectscandidate compounds which increase the transcription or translation ofthe CRF₂R or which increase the stability of the mRNA or CRF₂R protein.

[0128] In another embodiment, this invention provides methods forscreening one or more test compounds to identify candidate compoundswhich regulate the expression of the CRF or a CRF analog. Such assaysare performed essentially as described above for the assays to identifycandidate compounds which regulate expression of CRFRs with thefollowing modifications. To identify candidate compound which regulatetranscription from the CRF gene or a CRF analog gene, the reporter geneis operably associated with the regulatory region of the CRF gene or CRFanalog gene of interest and the source of cellular factors should befrom a cell type that expresses the gene of interest.

[0129] VII. Screening of Candidate Compounds Using Models of SkeletalMuscle Atrophy

[0130] Candidate compounds selected from one or more test compounds byan in vitro assay, as described above, can be further tested for theirability to regulate skeletal muscle mass or function in model systems ofskeletal muscle atrophy and/or hypertrophy. Such models of skeletalmuscle atrophy or hypertrophy include both in vitro cell culture modelsand in vivo animal models of skeletal muscle atrophy. Such additionallevels of screening are useful to further narrow the range of candidatecompounds that merit additional investigation, e.g., clinical trials.

[0131] Cell Culture Models of Muscle Atrophy

[0132] In vitro models of skeletal muscle atrophy are known in the art.Such models are described, for example, in Vandenburgh, H. H., In Vitro24:609-619 (1988), Vandenburgh, H. H. et al., J of Biomechanics, 24Suppl 1:91-99 (1991), Vandenburgh, H. H et al., In Vitro Cell. Dev.Biol., 24(3):166-174 (1988), Chromiak, J. A., et al., In Vitro Cell.Dev. Biol. Anim., 34(9):694-703 (1998), Shansky, J., et al., In VitroCell. Dev. Biol. Anim., 33(9):659-661 (1997), Perrone, C. E. et al., J.Biol. Chem. 270(5):2099-2106 (1995), Chromiac, J. A. and Vandenburgh, H.H., J. Cell. Physiol. 159(3):407-414 (1994), and Vandenburgh, H. H. andKarlisch, P., In Vitro Cell. Dev. Biol. 25(7):607-616 (1989). Suchmodels are useful, but not required, following the in vitro screeningdescribed above in order to further narrow the range of candidatecompounds that merit testing in an animal model. Cell culture models aretreated with candidate compounds and the response of the model to thetreatment is measured by assessing changes in muscle markers such as:muscle protein synthesis or degradation, changes in skeletal muscle massor contractile function. Those compounds which induce significantchanges in the muscle markers are typically screened further in ananimal model of skeletal muscle atrophy.

[0133] Animal Models of Skeletal Muscle Atrophy

[0134] The candidate compounds are administered to non-human animals andthe response of the animals is monitored, for example, by assessingchanges in markers of atrophy or hypertrophy such as: skeletal musclemass, skeletal muscle function, muscle or myofiber cross-sectional area,contractile protein content, non-contractile protein content or abiochemical or genetic marker that correlates with skeletal muscle massor function changes. Candidate compounds which induce skeletal musclehypertrophy or prevent any aspect of skeletal muscle atrophy should beconsidered as prospective therapeutic candidates for treatment of humanskeletal muscle atrophy, and are referred to herein as candidatetherapeutic compounds. In addition to assessing the ability of acandidate compound to regulate skeletal muscle atrophy, undesirable sideeffects such as toxicity may also be detected in such a screen. Theabsence of unacceptably high levels of side effects may be used as afurther criterion for the selection of candidate therapeutic compounds.

[0135] A variety of animal models for skeletal muscle atrophy are knownin the art, such as those described in the following references:Herbison, G. J., et al. Arch. Phys. Med. Rehabil. 60:401-404 (1979),Appell, H-J. Sports Medicine 10:42-58 (1990), Hasselgren, P-O. andFischer, J. E. World J. Surg. 22:203-208 (1998), Agbenyega, E. T. andWareham, A. C. Comp. Biochem. Physiol. 102A:141-145 (1992), Thomason, D.B. and Booth, F. W. J. Appl. Physiol. 68:1-12 (1990), Fitts, R. H., etal. J. Appl. Physiol. 60:1946-1953 (1986), Bramanti, P., et al. Int. J.Anat. Embryol. 103:45-64 (1998), Cartee, G. D. J. Gerontol. A Biol. Sci.Med. Sci. 50:137-141 (1995), Cork, L. C., et al. Prog. Clin. Biol. Res.229:241-269 (1987), Booth, F. W. and Gollnick, P. D. Med. Sci. SportsExerc. 15:415-420 (1983), Bloomfield, S. A. Med. Sci. Sports Exerc.29:197-206 (1997). Preferred animals for these models are mice and rats.These models include, for example, models of disuse-induced atrophy suchas casting or otherwise immobilizing limbs, hind limb suspension,complete animal immobilization, and reduced gravity situations. Modelsof nerve damage induced atrophy include, for example, nerve crush,removal of sections of nerves which innervate specific muscles, toxinapplication to nerves and infection of nerves with viral, bacterial oreukaryotic infectious agents. Models of glucocorticoid-induced atrophyinclude application of atrophy-inducing doses of exogenousglucocorticoid to animals, and stimulation of endogenous corticosteroidproduction, for example, by application of hormones that activate thehypothalamus-pituitary-adrenal (HPA) axis. Models of sepsis-inducedatrophy include, for example, inoculation with sepsis-inducing organismssuch as bacteria, treatment of the animal with immune-activatingcompounds such as bacterial cell wall extract or endotoxin, and punctureof intestinal walls. Models of cachexia-induced atrophy include, forexample, inoculation of an animal with tumorigenic cells with cachexiaforming potential, infection of an animal with infectious agents (suchas viruses which cause AIDS) which result in cachexia and treatment ofan animal with hormones or cytokines such as CNTF, TNF, IL-6, IL-1, etc.which induce cachexia. Models of heart failure-induced atrophy includethe manipulation of an animal so that heart failure occurs withconcomitant skeletal muscle atrophy. Neurodegenerative disease-inducedatrophy models include autoimmune animal models such as those resultingfrom immunization of an animal with neuronal components. Musculardystrophy-induced models of atrophy include natural or man-madegenetically-induced models of muscular dystrophy such as the mutation ofthe dystrophin gene which occurs in the Mdx mouse.

[0136] Animal models of skeletal muscle hypertrophy include, forexample, models of increased limb muscle use due to inactivation of theopposing limb, reweighting following a disuse atrophy inducing event,reutilization of a muscle which atrophied because of transient nervedamage, increased use of selective muscles due to inactivation of asynergistic muscle (e.g., compensatory hypertrophy), increased muscleutilization due to increased load placed on the muscle and hypertrophyresulting from removal of the glucocorticoid afterglucocorticoid-induced atrophy. Preferred animal atrophy models includethe sciatic nerve denervation atrophy model, glucocorticoid-inducedatrophy model, and the leg casting disuse atrophy model that aredescribed in further detail below.

[0137] The sciatic nerve denervation atrophy model involvesanesthetizing the animal followed by the surgical removal of a shortsegment of either the right or left sciatic nerve, e.g., in mice thesciatic nerve is isolated approximately at the midpoint along the femurand a 3-5 mm segment is removed. This denervates the lower hind limbmusculature resulting in atrophy of these muscles. Typically,innervation to the biceps femoris is left intact to provide satisfactorymotion of the knee for virtually normal ambulation. Typically, inuntreated animals, muscle mass of the denervated muscles is reduced30-50% ten days following denervation. Following denervation, testcompounds are administered e.g., by injection or by continuous infusion,e.g., via implantation of an osmotic minipump (e.g., Alzet, Palo Alto,Calif.), to determine their effect on denervation induced skeletalmuscle atrophy. At various times following denervation, the animals areeuthanized and lower leg muscles are dissected rapidly from both thedenervated and nondenervated legs, the muscles, cleaned of tendons andconnective tissue, are weighed. The extent of atrophy in the affectedmuscles is analyzed, for example, by measuring muscle mass, musclecross-sectional area, myofiber cross-sectional area or contractileprotein content.

[0138] The glucocorticoid-induced atrophy model involves theadministration of a glucocorticoid to the test animal, e.g., 1.2mg/kg/day of dexamethasone in the drinking water. Typically, inuntreated animals, skeletal muscle mass is reduced 30-50% following tendays of dexamethasone administration. Concomitantly with, or followingglucocorticoid administration, test compounds are administered e.g., byinjection or by continuous infusion to determine their effect onglucocorticoid-induced skeletal muscle atrophy. At various timesfollowing glucocorticoid administration, the extent of atrophy in theaffected muscles is analyzed as described above for the denervationmodel.

[0139] The leg casting disuse atrophy model involves casting one hindleg of an animal from the knee down through the foot. Typically, musclemass is reduced 20-40% after ten days of casting. Following casting,test compounds are administered by injection or by continuous infusionvia implantation of an osmotic minipump (e.g., Alzet, Palo Alto, Calif.)to determine their effect on leg casting induced skeletal muscleatrophy. At various times following leg casting, the extent of atrophyin the affected muscles is analyzed as described above for thedenervation model.

[0140] One of skill in the art would recognize that in screening forcompounds for human use, because there are differences between the humanCRF₂R and the CRF₂R from other animal species, there may be some falsepositive or negative results which arise when the screen is carried outusing non-human CRF₂R. Thus, it is preferable to do the initial in vitroscreen using human CRF₂R. In certain circumstances, identified candidatecompounds may be active toward only the human receptor and not toward anon-human receptor. In such circumstances, it may still be desirable todetermine whether these candidate compounds are able to regulateskeletal muscle mass or function in a second level of screening. Becausethese candidates do not activate non-human CRF₂R, a standard in vivoscreen with non-human animal is not advised. In such circumstances thesecond level of screening for these candidates may be performed intransgenic animals that express human CRFRs.

[0141] Animals of any species, especially mammals, including, but notlimited to, mice, rats, rabbits, guinea pigs, pigs, goats, dogs andnon-human primates may be used to generate CRFR transgenic animals. Miceand rats are preferred, mice are most preferred. A variety of techniquesare known in the art and may be used to introduce the human CRFRtransgenes into animals to produce the founder lines of transgenicanimals. Such techniques include, but are not limited to, pronuclearmicroinjection, retrovirus-mediated gene transfer into germ lines, genetargeting in embryonic stem cells, electroporation of embryos andsperm-mediated gene transfer.

[0142] VIII. Gene Therapy Methods for the Treatment of Skeletal MuscleAtrophy

[0143] The overall activity of CRF₂R can be increased by overexpressinga gene for CRF₂R (to increase expression of CRF₂R) or a constitutivelyactive CRF₂R in the appropriate tissue. CRF levels can be increased, invivo, by likewise overexpressing a CRF gene. Overexpression of thesegenes will increase the total cellular CRF₂R activity, thus, regulatingskeletal muscle atrophy. The gene or genes of interest are inserted intoa vector suitable for expression in the subject. These vectors include,but are not limited to, adenovirus, adenovirus associated virus,retrovirus and herpes virus vectors in addition to other particles thatintroduced DNA into cells (e.g., liposome, gold particles, etc.) or bydirect injection of the DNA expression vector, containing the gene ofinterest, into human tissue (e.g., muscle).

[0144] IX. Pharmaceutical Formulations and Methods for Use

[0145] Candidate compounds or candidate therapeutic compounds identifiedby screening methods described herein, can be administered toindividuals to treat skeletal muscle atrophy, or to induce skeletalmuscle hypertrophy. To this end, the present invention encompassesmethods and compositions for modulating skeletal muscle atrophy,including, but not limited to, skeletal muscle atrophy induced by disusedue to surgery, bed rest, broken bones; denervation/nerve damage due tospinal cord injury; autoimmune disease; infectious disease;glucocorticoid use for unrelated conditions; sepsis due to infection orother causes; nutrient limitation due to illness or starvation; cancercachexia; chronic inflammation; AIDS cachexia; COPD; congestive heartfailure; sarcopenia and genetic disorders; e.g., muscular dystrophies,neurodegenerative diseases. Agonists of CRF₂R can be used to inhibitskeletal muscle atrophy. It is not necessary that effective compoundsdemonstrate absolute specificity for CRFR. It is contemplated thatspecific antagonist of other affected receptors can be co-administeredwith an effective, but nonspecific, agonist. Alternately, this lack ofspecificity may be addressed by modulation of dose alone, or the dosingregimen.

[0146] The candidate compounds or candidate therapeutic compoundsidentified by the screening methods of the present invention may beadministered in conjunction with compounds which prolong or augment theactivation of a CRF₂R or of a CRF₂R signal transduction pathway. Thesemay be known compounds, for example, theophylline, or these compoundsmay be identified by the screening methods of this invention to prolongor augment the activation of a CRF₂R receptor or of a CRF₂R signaltransduction pathway.

[0147] Dose Determinations

[0148] Safety and therapeutic efficacy of compounds which agonize CRFRcan be determined by standard procedures using either in vitro or invivo technologies. Compounds which exhibit large therapeutic indices arepreferred, although compounds with lower therapeutic indices are usefulif the level of side effects is acceptable. The data obtained from thein vitro and in vivo toxicological and pharmacological techniques can beused to formulate the human range of doses which may be useful. Thepreferred dose lies in the range in which the circulating concentrationof the compound is therapeutically maximal with acceptable safety. Thecirculating concentration of the compound may vary depending on the doseform, time after dosing, route of administration, etc. Doses outsidethis range are also useful provided the side effects are acceptable.Such matters as age and weight of the patient, and the like, can be usedto determine such matters in the conventional manner. Pharmacogeneticapproaches may be useful in optimizing compound selection, doses anddosing regimen in clinical populations.

[0149] Formulation and Use

[0150] Pharmaceutical compositions for use in the modulation of skeletalmuscle atrophy in accordance with the present invention may beformulated using conventional methodologies using pharmaceuticallyacceptable carriers and excipients. The compositions of this inventionare preferably provided in unit dosage form. As used herein, a “unitdosage form” is a composition of this invention containing an amount ofa CRF₂R agonist that is suitable for administration to an animal,preferably a mammal, more preferably a human subject, in a single dose,according to good medical practice. Pharmaceutical compositions may beformulated for delivery by, for example, intranasal, transdermal,inhalation, parenteral, cutaneous, oral or rectal administration. Fororal administration, the pharmaceutical composition may take the form oftablets or capsules containing the pharmacologically active compound andadditives including, but not limited to, binding agents, fillers,lubricants, disintegrants, or wetting agents. The tablets may be coated.Liquid preparations for oral administration include, but are not limitedto, syrups, suspensions or dry products which are reconstituted withliquid vehicle before use, containing the pharmacologically activecompound and additives including, but not limited to, suspending agents,emulsifying agents, non-aqueous vehicles, preservatives, buffer salts,flavoring, coloring, sweetening agents, etc. Pharmaceutical compositionsfor oral administration may be formulated for controlled release of thepharmacologically active compounds either in the mouth, stomach orintestinal tract.

[0151] For inhalation administration, the compounds for use according tothe present invention may be delivered by, but not limited to, thefollowing forms: liquid, powder, gel or in the form of an aerosol sprayutilizing either pressurized or non-pressurized propellants in eitherpremeasured or non-premeasured doses. The pharmacologically activecompound may be formulated with appropriate fillers, vehicles,preservatives, buffers, etc. For parenteral administration, thepharmacologically active compound may be formulated with acceptablephysiological carriers, preservatives, etc. and be prepared assuspensions, solutions, emulsion, powders ready for constitution, etc.for either bolus injection or infusion. Doses of these compounds may beadministered by a variety of technologies including hypodermic needles,high pressure devices, etc. For rectal administration, thepharmacologically active compound may be formulated with acceptablephysiological carriers, preservatives, etc. for delivery assuppositories, enemas, etc. For cutaneous administration, thepharmacologically active compound may be formulated with acceptablephysiological carriers including lotions, emollients, etc. orincorporated into a patch type device. For long term administration, thepharmacologically active compound and appropriate additives such as, butlimited to, polymers, hydrophobic materials, resins, etc. may beformulated as a depot preparation for either injection or implantationat multiple sites including but not limited to intramuscular andsubcutaneous locations. In addition, the pharmacologically activecompound may be administered by a dispensing device.

[0152] Monitoring of Effects During Clinical Trials

[0153] Monitoring the influence of compounds (e.g., drugs) on theexpression or activity of CRF₂R can be employed not only in basic drugscreening, but also in clinical trials. For example, the effectivenessof a compound determined by a screening assay to increase CRF₂R receptoractivity or CRF₂R receptor expression can be assessed in clinical trialsof patients with, or at risk for, skeletal muscle atrophy. At varioustimes following administration of the test compound or placebo, theeffect of the compound on the patient can be determined, for example, byobserving the change in skeletal muscle mass, skeletal muscle function,biochemical markers of muscle breakdown or quality of life measures.Methods of measuring skeletal muscle mass in human subjects are known inthe art and include, for example: measuring the girth of a limb;measuring muscle thickness with for instance, computer tomography, MRIor supersonics; or muscle biopsy to examine morphological andbiochemical parameters (e.g., cross-section fiber area, fiber diameteror enzyme activities). Furthermore, because skeletal muscle mass iscorrelated with skeletal muscle function, muscle function can be used asa surrogate marker of mass and muscle mass changes can be assessed usingfunctional measurements, e.g., strength, the force of a group ofsynergist muscles, or contraction characteristics found inelectromyographic recordings. In addition, muscle protein loss as aresult of muscle atrophy can be measured by quantitating levels of aminoacids or amino acids derivatives, i.e., 3-methyl histidine, in the urineor blood of a subject. For a review of such methods see Appell, SportsMed. 10:42-58 (1990). Quality of life measures include, but are notlimited to, the ease of getting out of a chair, number of steps takenbefore tiring or ability to climb stairs.

EXAMPLES Example 1

[0154] Construction of Vectors for Human CRF₂R Receptor Expression.

[0155] The human CRF₂R (hCRF₂R) DNA sequence, Accession No. E12752, isretrieved and two oligonucleotides including one containing the 5′ endof the gene beginning at the initiation codon (5′ oligonucleotide) andone containing the 3′ end of the gene containing the stop codon (3′oligonucleotide) are synthesized. These oligonucleotides are designed tocontain restriction endonuclease sites which are not present in thehCRF₂R gene with one unique site in the 5′ oligonucleotide and adifferent unique restriction endonuclease site in the 3′ oligonucleotideIn addition, the 3′ oligonucleotide contains a polyadenylation additionsignal sequence. Double stranded cDNA from human skeletal muscle ispurchased from the Universal QUICK-Clone cDNA collection (ClonetechInc., Palo Alto, Calif., USA). Using the above 5′ and 3′oligonucleotides, the hCRF₂R cDNA is amplified by PCR of the humanskeletal muscle cDNA using the AdvanTaq PCR kit (Clonetech Inc., PaloAlto, Calif., USA). The hCRF₂R gene PCR product is purified from PCRartifacts by agarose gel electrophoresis and the hCRF₂R gene DNAfragment is purified from the agarose gel using a purification productsuch as NucleoTrap (Clonetech Inc., Palo Alto, Calif., USA).

[0156] Cloning of the hCRF₂R PCR product into the pIRESneo vector(Clonetech Inc., Palo Alto, Calif., USA) is accomplished by firstcutting the hCRF₂R PCR product and the pIRESneo vector with theappropriate restriction endonucleases so that the 5′ and 3′ restrictionendonuclease sites are ready for ligation. The pIRESneo vector DNA isligated to the hCRF₂R PCR product DNA using DNA ligase, from theAdvantAge™PCR Cloning Kit (Clonetech Inc., Palo Alto, Calif., USA),according to the manufacturer's recommendations. The ligated vector andinsert construct (pIRESneo/hCRF₂R)is then used to transform TOP10F′competent E. coli cells (Clonetech Inc., Palo Alto, Calif., USA).Transformed cells are plated on LB/X-gal/IPTG plus ampicillin containingagar. White colonies (positive clones) are selected and individuallycultured in LB medium. Plasmid DNA is isolated using NucleoBond DNAPurification System (Clonetech Inc., Palo Alto, Calif., USA). The insertfrom at least one clone is sequenced to ensure that the hCRF₂R sequenceis correct. HEK293 cells containing a stably integrated Mercury CRE-LUCplasmid (Clonetech Inc., Palo Alto, Calif., USA) are transfected withpurified pIRESneo/hCRF₂R DNA, having the correct sequence insert,utilizing the CalPhos™ Mammalian Transfection Kit (Clonetech Inc., PaloAlto, Calif., USA. Cells stably transfected with pIRESneo/hCRF₂R DNA areselected by culturing the cells in G418. The stably transfected cells(HEK293/CRE-LUC/pIRESneo/hCRF₂R cells) are propagated in DMEM (LifeTechnologies, Rockville, Md.) containing 10% fetal bovine serum(Clonetech Inc., Palo Alto, Calif., USA), penicillin/streptomycinsolution (Life Technologies, Rockville, Md.), L-glutamine (LifeTechnologies, Rockville, Md.), and non-essential amino acid (LifeTechnologies, Rockville, Md.) at 37° C. in a 5% carbon dioxide/95% airatmosphere. The clones are characterized for both CRF binding andCRE-LUC activation following exposure to CRF as described in Example 2and Example 3. Cells expressing the hCRF₂R receptor at an appropriatelevel and which are appropriately coupled to the CRE-LUC reporter systemare then utilized for further analysis.

Example 2

[0157] Receptor Binding Assays

[0158] Receptor binding analysis of compounds is performed in wholecells by plating the HEK293/CRE-LUC/pIRESneo/hCRF₂R cells from Example 1in a 96 well polylysine coated plate. Cells are seeded in DMEM mediumcontaining 10% fetal bovine serum, penicillin/streptomycin solution,L-glutamine, and non-essential amino acid at 37° C. in a 5% carbondioxide/95% air atmosphere and incubated overnight. The culture mediumis removed and the appropriate amount of CRF covalently labeled withEuropium (Eu-CRF) in MEM (Life Technologies, Rockville, Md.)+10%Seablock (Clonetech Inc., Palo Alto, Calif., USA) is added. The cellsare incubated with the Eu-CRF for 90 minutes at room temperature thenwashed 4 times with phosphate buffered saline lacking magnesium andcalcium (Life Technologies, Rockville, Md.). Following the final wash,enhancement solution is added (Wallac Inc., Gaithersburg, Md.) and theplate is read on a Wallac plate reader (Wallac Inc., Gaithersburg, Md.)using the BioWorks Europium program. For saturation binding analysis,log doses of Eu-CRF ranging from 10(-12) to 10(-3) M are added to thecells and binding analyzed both in the absence and the presence of asaturating concentration of unlabeled CRF for evaluation of non-specificbinding. For competitive binding, a concentration of Eu-CRF is addedwhich is half maximal, in terms of binding, in addition to varyingconcentrations of the compound of interest.

Example 3

[0159] Receptor Activation Assay

[0160] Receptor activation analysis is performed by seeding theHEK293/CRE-LUC/pIRESneo/hCRF₂R cells of Example 1 into Packard ViewPlate-96 (Packard Inc., CA). Cells are seeded in DMEM medium containing10% fetal bovine serum, penicillin/streptomycin solution, L-glutamine,and non-essential amino acid at 37° C. in a 5% carbon dioxide/95% airatmosphere and incubated overnight. The medium is then removed andreplaced with DMEM (Life Technologies, Rockville, Md.) containing 0.01%bovine albumin fraction V (SIGMA, St. Louis, Mo.) containing thecompound of interest. The cells are then incubated for four hours at 37°C. in a 5% carbon dioxide/95% air atmosphere after which the medium isremoved and the cells are washed twice with Hanks Balanced Salt Solution(Life Technologies, Rockville, Md.). Lysis Reagent (Promega Inc.,Madison, Wis.) is then added to the washed cells and the cells areincubated for 20 minutes at 37° C. in a 5% carbon dioxide/95% airatmosphere. The cells are then placed at −80° C. for 20 minutes followedby a 20 minute incubation at 37° C. in a 5% carbon dioxide/95% airatmosphere. After this incubation, Luciferase Assay Buffer andLuciferase Assay Substrate (Promega Inc., Madison, Wis.) are added tothe cell lysates and luciferase activity quantitated using aluminometer. Relative activity of a compound is evaluated by comparingthe increase following exposure to compound to the level of luciferasein HEK cells which contain the CRE-LUC construct without the hCRF₂Rfollowing exposure to compound. Specificity of response is also checkedby evaluating luciferase response of hCRF₂R/CRE-LUC HEK cells tocompound in the presence and absence of a 10-fold excess of hCRF₂Rantagonist.

Example 4

[0161] Screen to Identify Candidate Compounds that Prolong or Augmentthe Activation of CRF₂R and/or a CRF₂R Receptor Signal TransductionPathway.

[0162] Identification of compounds that prolong or augment theagonist-induced activation of the CRF₂R or of a CRF₂R signaltransduction pathway, involves a variation of the Receptor ActivationAssay described in Example 3. Specifically, this assay is performed byseeding the HEK293/CRE-LUC/pIRESneo/hCRF₂R receptor cells into PackardView Plate-96 (Packard Inc., CA). Cells are seeded in DMEM mediumcontaining 10% fetal bovine serum, penicillin/streptomycin solution,L-glutamine, non-essential amino acid, and saturating amounts of CRF at37° C. in a 5% carbon dioxide/95% air atmosphere and incubated for 48hours. The medium is then removed and replaced with DMEM (LifeTechnologies, Rockville, Md.) containing 0.01% bovine albumin fraction V(SIGMA, St. Louis, Mo.) and CRF in addition to the compound of interest.The cells are then incubated for four hours at 37° C. in a 5% carbondioxide/95% air atmosphere after which the medium is removed and thecells are washed twice with Hanks Balanced Salt Solution (LifeTechnologies, Rockville, Md.). Lysis Reagent (Promega Inc., Madison,Wis.) is then added to the washed cells and the cells are incubated for20 minutes at 37° C. in a 5% carbon dioxide/95% air atmosphere. Thecells are then placed at −80° C. for 20 minutes followed by a 20 minuteincubation at 37° C. in a 5% carbon dioxide/95% air atmosphere. Afterthis incubation, Luciferase Assay Buffer and Luciferase Assay Substrate(Promega Inc., Madison, Wis.) are added to the cell lysates andluciferase activity is quantitated using a luminometer. Test compoundswhich stimulate fluorescence significantly above the levels of controluntreated cells, after correction for variations in cell density, areconsidered candidate compounds for regulating skeletal muscle mass orfunction. The compounds of most interest are those that inducerelatively higher levels of fluorescence.

Example 5

[0163] Screen to Identify Candidate Compounds Specific for CRF₂R.

[0164] Compounds that activate CRF₂R are identified as in Example 3. Toselect those compounds which show selectivity for CRF₂R over CRF₁R,these compounds also are screened against CRF₁R.HEK293/CRE-LUC/pIRESneo/hCRF₁R cells are generated essentially asdescribed in Example 1 except that the human CRF₁R (hCRF₁R) DNAsequence, Accession No. X72304, is used for the initial PCRamplification. To determine how active the compounds are against CRF₁R,an activation assay is performed essentially as described in Example 3except that HEK293/CRE-LUC/pIRESneo/hCRF₁R cells are used to seed theplates. The amount of fluorescence stimulated by the compound in CRF₂Rexpressing cells is compared with the amount of fluorescence stimulatedby the compound in CRF₁R expressing cells. Those compounds whichdemonstrate a 10-fold better response (on a molar basis) in CRF₂Rexpressing cells than in CRF₁R expressing cells are then checked furtherfor specificity of response to eliminate differences due to clonalvariation. HEK293/CRE-LUC/pIRESneo/hCRF₂R cells are assayed with thecompound in the presence or absence of a 10-fold excess of the CRF₂Rantagonist, antisauvagine-30. Those compounds that show greater than10-fold selectivity for CRF₂R and whose activity is inhibited byantisauvagine-30 are selected as candidate compounds.

Example 6

[0165] Screens to Identify Candidate Compounds that Increase hCRF₂RExpression

[0166] The sequence containing the promoter region of the hCRF₂R gene,beginning far enough upstream of the transcriptional initiation site tocontain all the regulatory elements necessary for physiologicalexpression of the hCRF₂R gene in the appropriate tissue is retrievedfrom the human genome database. Two oligonucleotides, one containing the5′ end of the promoter region (5′ oligonucleotide) and one containingthe 3′ end of the promoter region including the transcriptional startsite (3′ oligonucleotide) are synthesized. These oligonucleotides alsocontain restriction endonuclease sites which are not present in thehCRF₂R gene regulatory region with one unique site in the 5′oligonucleotide and a different unique restriction endonuclease site inthe 3′ oligonucleotide. The 5′ and 3′ oligonucleotides are used for PCRamplification of the hCRF₂R gene regulatory region from human DNA(Clonetech Inc., Palo Alto, Calif., USA) using the PCR kit,Advantage®Genomic PCR kit (Clonetech Inc., Palo Alto, Calif., USA). ThehCRF₂R gene regulatory region PCR product is purified from PCR artifactsby agarose gel electrophoresis and the hCRF₂R gene regulatory region DNAfragment is purified from the agarose gel using a purification productsuch as NucleoTrap (Clonetech Inc., Palo Alto, Calif., USA). Cloning ofthe hCRF₂R gene regulatory region PCR product into the pECFP-1 vector(Clonetech Inc., Palo Alto, Calif., USA) is accomplished by firstcutting the hCRF₂R gene regulatory region PCR product and the pECFP-1vector with the appropriate restriction endonucleases so that the 5′ and3′ restriction endonuclease sites are ready for ligation. Ligation ofthe pECFP-1 vector DNA to the hCRF₂R gene regulatory region PCR productDNA is accomplished using DNA ligase from the AdvantAge™PCR Cloning Kit(Clonetech Inc., Palo Alto, Calif., USA) according to the manufacturer'srecommendations. The ligated vector and insert construct is then used totransform TOP10F′ competent E. coli cells (Clonetech Inc., Palo Alto,Calif., USA). The cells are plated on LB plus kanamycin containing agarand kanamycin resistant colonies are selected for further analysis.Kanamycin resistant clones are cultured in LB containing kanamycinmedium and plasmid DNA is isolated using NucleoBond DNA PurificationSystem (Clonetech Inc., Palo Alto, Calif., USA) and the constructcontaining the hVPAC₂ gene regulatory region is analyzed by DNAsequencing to ensure construct correctness and integrity. Purifiedconstruct plasmid DNA containing the hCRF₂R gene regulatory region isthen transfected into the HEK293 cells utilizing calciumphosphate-mediated transfection utilizing the CalPhos™ MammalianTransfection Kit (Clonetech Inc., Palo Alto, Calif., USA). Transfectedcell clones are selected using G418, isolated and propagated in DMEM(Life Technologies, Rockville, Md.) containing 10% fetal bovine serum(Clonetech Inc., Palo Alto, Calif., USA), penicillin/streptomycinsolution (Life Technologies, Rockville, Md.), L-glutamine (LifeTechnologies, Rockville, Md.), non-essential amino acid (LifeTechnologies, Rockville, Md.) and G418 (Life Technologies, Rockville,Md.) at 37° C. in a 5% carbon dioxide/95% air atmosphere. G418 resistantclones are characterized by Southern blotting to ensure that theycontain the hCRF₂R gene promoter sequence; in addition activation of thehCRF₂R gene regulatory region is analyzed using an appropriatestimulating agent. Cells expressing the hCRF₂R gene regulatoryregion-ECFP at an appropriate level are then used in assays designed toevaluate compounds which can modulate the activity of the hCRF₂R generegulatory region as follows. The regulatory region activation analysisis performed by seeding the hCRF₂R gene regulatory region-ECFPcontaining HEK293 cells at an appropriate density into black with clearbottom 96 well microtiter plates and allowed to grow overnight. Thefollowing day, the medium is removed and the test compound added infresh growth medium. The cells are incubated for 16 hours at 37° C. in a5% carbon dioxide/95% air atmosphere followed by measurement offluorescence (excitation at 433 (453) nm by detecting emission at475(501) nm using a fluorometer (biolumin™ 960, MolecularDynamics/Amersham Pharmacia Biotech, Piscataway, N.J.). Test compoundswhich stimulate fluorescence significantly above the levels of controluntreated cells, after correction for variations in cell density, areconsidered candidate compounds for regulating skeletal muscle mass orfunction. The compounds of most interest are those which inducerelatively higher levels of fluorescence.

Example 7

[0167] Screens to Identify Compounds that Increase Human CRF Expression

[0168] The methods for identifying compounds that increase human CRF(hCRF) expression are essentially identical to those for identifyingcompounds which increase hVPAC₂ receptor expression except theregulatory region used is that for the hCRF gene. The sequencecontaining the regulatory region of the hCRF gene, beginning far enoughupstream of the transcriptional initiation site to contain all theregulatory elements necessary for physiological expression of the hCRFgene in the appropriate tissue is retrieved from the human genomedatabase. Two oligonucleotides, one containing the 5′ end of theregulatory region (5′ oligonucleotide) and one containing the 3′ end ofthe regulatory region including the transcriptional start site (3′oligonucleotide) are synthesized. These oligonucleotides also containrestriction endonuclease sites which are not present in the hCRF generegulatory region with one unique site in the 5′ oligonucleotide and adifferent unique restriction endonuclease site in the 3′oligonucleotide. The 5′ and 3′ oligonucleotides are used for PCRamplification of the hCRF gene regulatory region from human DNA(Clonetech Inc., Palo Alto, Calif., USA) using the Advantage®Genomic PCRkit (Clonetech Inc., Palo Alto, Calif., USA). The hCRF gene regulatoryregion PCR product is purified from PCR artifacts by agarose gelelectrophoresis and the hCRF gene regulatory region DNA fragment ispurified from the agarose gel using the purification product, NucleoTrap(Clonetech Inc., Palo Alto, Calif., USA). Cloning of the hCRF generegulatory region PCR product into the pECFP-1 vector (Clonetech Inc.,Palo Alto, Calif., USA) is accomplished by first cutting the hCRF generegulatory region PCR product and the pECFP-1 vector with theappropriate restriction endonucleases so that the 5′ and 3′ restrictionendonuclease sites are ready for ligation. Ligation of the pECFP-1vector DNA to the hCRF gene regulatory region PCR product DNA isaccomplished using DNA ligase from AdvantAge™PCR Cloning Kit (ClonetechInc., Palo Alto, Calif., USA) according to the manufacturer'srecommendations. The ligated vector and insert construct is then used totransform TOP10F′ competent E. coli cells (Clonetech Inc., Palo Alto,Calif., USA). The cells are plated on LB plus kanamycin containing agarand kanamycin resistant colonies are selected for further analysis.Kanamycin resistant clones are cultured in LB containing kanamycinmedium and plasmid DNA is isolated using NucleoBond DNA PurificationSystem (Clonetech Inc., Palo Alto, Calif., USA) and the constructcontaining the hCRF gene regulatory region is analyzed by DNA sequencingto ensure construct correctness and integrity. Purified constructplasmid DNA containing the hCRF gene regulatory region is thentransfected into the HEK293 cells utilizing calcium phosphate-mediatedtransfection utilizing the CalPhos™ Mammalian Transfection Kit(Clonetech Inc., Palo Alto, Calif., USA). Transfected cell clones areselected using G418, isolated and propagated in DMEM (Life Technologies,Rockville, Md.) containing 10% fetal bovine serum (Clonetech Inc., PaloAlto, Calif., USA), penicillin/streptomycin solution (Life Technologies,Rockville, Md.), L-glutamine (Life Technologies, Rockville, Md.),non-essential amino acid (Life Technologies, Rockville, Md.) and G418(Life Technologies, Rockville, Md.) at 37° C. in a 5% carbon dioxide/95%air atmosphere. G418 resistant clones are characterized by Southernblotting to ensure that they contain the hCRF gene regulatory regionsequence; in addition activation of the hCRF gene regulatory region isanalyzed using an appropriate stimulating agent. Cells expressing thehCRF gene regulatory region-ECFP at an appropriate level are then usedin assays designed to evaluate compounds which can modulate the activityof the hCRF gene regulatory region as follows. The regulatory regionactivation analysis is performed as in Example 5 except that clonescontaining the hCRF gene regulatory region construct are used.

Example 8

[0169] Method of Making Human Antibodies which Activate the hCRF₂R.

[0170] Fully human monoclonal antibodies which activate the hCRF₂R areproduced by first generating recombinant hCRF₂R protein as follows. Theprocedure from Example 1 is followed to obtain the hCRF₂R PCR product.This hCRF₂R PCR product is then cloned into the pHAT20 vector (ClonetechInc., Palo Alto, Calif., USA) by first cutting the hCRF₂R gene PCRproduct and the pHAT20 vector with the appropriate restrictionendonucleases so that the 5′ and 3′ restriction endonuclease sites areready for ligation. Ligation of the pHAT20 vector DNA to the hCRF₂R genePCR product DNA is accomplished using DNA ligase from the AdvantAge™PCRCloning Kit (Clonetech Inc., Palo Alto, Calif., USA) according to themanufacturer's recommendations. The ligated vector/insert construct isthen used to transform TOP10F′ competent E. coli cells (Clonetech Inc.,Palo Alto, Calif., USA). Transformed cells are plated on LB plusampicillin containing agar and ampicillin resistant colonies areselected for further analysis. Positive clones are cultured in LB mediumcontaining ampicillin and plasmid DNA is isolated using NucleoBond DNAPurification System (Clonetech Inc., Palo Alto, Calif., USA) and theconstruct containing the hCRF₂R gene is analyzed by DNA sequencing theensure construct correctness and integrity. The hCRF₂R-pHAT20 vector DNAis then used for additional PCR cloning by utilizing a 5′oligonucleotide containing the beginning of the HAT sequence and aunique restriction endonuclease site not present in the hCRF₂R-pHAT20construct and the 3′ hCRF₂R oligonucleotide utilized previously. Theoligonucleotide primers are used to PCR amplify the HAT-hCRF₂R fusiongene from the hCRF₂R-pHAT20 construct and the PCR product is purified asdescribed above. The HAT-hCRF₂R fusion gene PCR product is then utilizedfor cloning into the pBacPAK8 vector using the BacPAK BaculovirusExpression System from Clonetech (Clonetech Inc., Palo Alto, Calif.,USA). The ligation of the HAT-hCRF₂R fusion gene into the pBacPAK8vector is essentially as described above. The hCRF₂R/HAT-pBacPAK8construct is then transfected into TOP10′F competent E. coli cells,ampicillin resistant cells are selected and plasmid DNA is isolated andchecked for construct integrity as described above. This construct isthen cotransfected with linearized BacPAK6 DNA into Sf21 insect hostcells utilizing the CalPhos™ Mammalian Transfection Kit (Clonetech Inc.,Palo Alto, Calif., USA). The insect cells are then incubated for 2-3days followed by harvest of virus from individual clear plaques. Thevirus is then amplified in Sf21 cells, the harvested virus titered, andthe titered virus used for large scale infection of Sf21 cells utilizingBacPAK Insect Cell Media—all according to the manufacturersrecommendations (Clonetech Inc., Palo Alto, Calif., USA). RecombinantHAT-CRF₂R fusion protein is then purified using the TALON® CellThruPurification Kit from Clonetech (Clonetech Inc., Palo Alto, Calif., USA)using conditions recommended by the manufacturer. Briefly, infected Sf21cells are harvested 48 hours after infection and sonicated inextraction/loading buffer. The cell lysate is then put through a TALON®CellThru column. The column is washed twice with extraction/loadingbuffer and the bound HAT-hCRF₂R protein is eluted with elution buffer.The eluted protein is analyzed by SDS-PAGE for integrity and proteinconcentration is quantitated using the Bio-Rad SDS-PAGE system andprotein quantitation systems according to the manufacturer'srecommendations (Bio-Rad Laboratories, Hercules, Calif.). PurifiedHAT-hCRF₂R fusion protein is then used for immunizing XenoMouse animals(Abgenix Inc., Fremont, Calif.) for human monoclonal antibody productionas follows. 10 μg of purified recombinant HAT-hCRF₂R fusion protein incombination with 25 μg of adjuvant monophosphoryl lipid A (Sigma, St.Louis, Mo.) is used to vaccinate 10 XenoMouse animals multiple timesover an eight week period. Serum is obtained from vaccinated animals andutilized in an antigen capture ELISA utilizing purified HAT-hCRF₂Rfusion protein to detect antibodies to the HAT-hCRF₂R protein by coatingpolystyrene ELISA plates (Corning Glass Works, Corning, N.Y.) withHAT-hCRF₂R fusion protein, blocked with PBS-1% BSA, washed and incubatedat 37° C. for 1 hour with a 1:50 dilution of the serum samples. Afterwashing 5 times with PBS, the plates are incubated at 37° C. for 1 hourwith alkaline phosphatase-conjugated goat antibodies to humanimmunoglobulin G. The plates are then washed 5× with PBS and antibodiesdetected with p-nitrophenyl phosphate substrate (Sigma, St. Louis, Mo.)in buffer. Optical densities at 405 nm were measured using a platereader and signal quantitated. Mice with demonstrated high antibodyproduction are used for hybridoma formation. Hybridomas are generated byfusion of splenic cells from the XenoMouse animals with nonsecretingmyeloma cell line NSA-bcl 2 using a 4:1 ratio of spleen cells toNSA-bcl2 cells in the presence of 30% polyethylene glycol PEG1450. Fusedcells are individually cloned by limiting dilution into 96 well platesand cultured in RPMI-1640 medium containing 10% fetal bovine serum,nonessential amino acids, sodium pyruvate, L-glutamine, 100 u/mlpenicillin-streptomycin and hypoxanthine-aminopterin-thymidine (all fromLife Technologies, Rockville, Md.). Supernatants from thehypoxanthine-aminopterin-thymidine selected hybridomas were screened forhuman antibody production by ELISA as described previously. Hybridomaswhich produce human antibodies to the HAT-hCRF₂R fusion protein areselected for large scale antibody production. Monoclonal antibodies arepurified by Protein G-Sepharose chromatography. Briefly, the supernatantfrom cultured hybridoma clones is loaded onto a Protein G-Sepharosecolumn (SIGMA, St. Louis, Mo.) in loading buffer, washed 3 times and theIgG is eluted with elution buffer. These antibodies are then used forscreening to evaluate hCRF₂R activation (agonism) potential. This isaccomplished using the methodology as outlined in Example 3. Those humanmonoclonal antibodies which demonstrate agonist activity toward thehCRF₂R are designated candidate compounds.

Example 9

[0171] Determination of Absolute Force Measurement of a Muscle.

[0172] The extensor digitorum longus (EDL) and soleus muscles areremoved, tendon-to-tendon from the casted mouse leg. A silk suture istied to each tendon of the isolated muscles and the muscles are placedinto a plexiglass chamber filled with Ringer solution (137 mM sodiumchloride, 24 mM sodium bicarbonate, 11 mM glucose, 5 mM potassiumchloride, 1 mM magnesium sulfate, 1 mM sodium phosphate, 0.025 mMtubocurarine, all at pH 7.4 and oxygenated with 95% oxygen/5% carbondioxide) constantly bubbled with 95% oxygen/5% carbon dioxide maintainedat 25° C. Muscles are aligned horizontally between a servomotor leverarm (Model 305B-LR Cambridge Technology Inc., Watertown Mass., USA) andthe stainless steel hook of a force transducer (Model BG-50; KuliteSemiconductor Products Inc., Leonia, N.J., USA) and field stimulated bypulses transmitted between two platinum electrodes placed longitudinallyon either side of the muscle. Square wave pulses (0.2 ms duration)generated by a personal computer with a Labview board (Model PCI-MIO16E-4), Labview Inc., Austin, Tex., USA) are amplified (Acurus poweramplifier model A25, Dobbs Ferry, N.Y., USA) to increase titaniccontraction. Stimulation voltage and muscle length (Lo) are adjusted toobtain maximum isometric twitch force. Maximum titanic force production(Po) is determined from the plateau of the frequency-force relationship.

Example 10

[0173] Therapeutic Treatment of Skeletal Muscle Atrophy Using a HumanAntibody that is an Agonist of the hCRF₂R Receptor.

[0174] A human male subject weighing 50 kg and having significantmuscular atrophy of the arms and legs due to prolonged bed rest, istreated to reverse the skeletal muscle atrophy. Once each week for aperiod of 3 months, 15 mls of an aqueous solution of pH 6 comprising anactivating antibody of the CRF₂R receptor is administered to the subjectvia intravenous injection. The solution comprises the following:Component Concentration (mg/ml) CRF₂R receptor agonist antibody 20L-histidine HCl 0.47 L-histidine 0.3 α, α-trehalose dihydrate 20Polysorbate 20 0.1 Bacteriostatic Sterile water qs to 1 mL

[0175] At the end of the treatment period, the subject exhibitsmeasurable increases of muscle mass, strength and mobility of the armsand legs.

Example 11

[0176] Prophylactic Treatment of Skeletal Muscle Atrophy Using a HumanAntibody that is an Agonist of the hCRF₂R Receptor.

[0177] A human female subject weighing 55 kg is scheduled for hip jointreplacement surgery in one month. The subject is treated to enhanceskeletal muscle mass prior to and following surgery to ultimately reducethe level of skeletal muscle atrophy due to muscle disuse duringpost-surgery recovery. Specifically, once each week for a period of 1month prior to surgery and for 2 months post-surgery, 18 ml of anaqueous solution of pH 6.0 comprising an activating antibody of theCRF₂R receptor, is administered to the subject via intravenousinjection. The solution comprises the following: Component Concentration(mg/ml) CRF₂R activating antibody 20 L-histidine HCl 0.47 L-histidine0.3 α, α-trehalose dihydrate 20 Polysorbate 20 0.1 BacteriostaticSterile water qs to 1 mL

[0178] At the end of the treatment period, the subject exhibitsmeasurable preservation of muscle mass, strength and mobility of thearms and legs as compared to the subject's expected status withoutantibody therapy.

Example 12

[0179] Prophylactic Treatment of Skeletal Muscle Atrophy Using a HumanAntibody that is an Agonist of the CRF₂R Receptor.

[0180] A human female subject weighing 45 kg undergoes a castingprocedure to treat a simple fracture of the humerus after a fall. Thesubject is treated to prevent atrophy of the skeletal muscle of theaffected arm and shoulder due to disuse and limited use during fracturehealing. Specifically, once each week starting on the day of casting, 13ml of pH 6.0 comprising the anti-hCRF₂R receptor is administered to thesubject via intravenous injection. The solution comprises the following:Component Concentration (mg/ml) CRFR activating antibody 20 L-histidineHCl 0.47 L-histidine 0.3 α, α-trehalose dihydrate 20 Polysorbate 20 0.1Bacteriostatic Sterile water qs to 1 mL

[0181] At the end of the treatment period, the subject exhibitsmeasurable preservation of muscle mass, strength and mobility of theaffected arm and shoulder and a reduced course of physical therapy ascompared to the subject's expected status and follow-up treatmentwithout antibody therapy.

Example 13

[0182] Prophylactic Treatment of Skeletal Muscle Atrophy UsingUrocortin-II

[0183] A human female subject weighing 60 kg is admitted to the hospitalin a comatose state. The subject is treated by this method to preventatrophy of the skeletal muscle of the entire body due to disuse in thecomatose state. Specifically, once each day while in the coma, thesubject is administered, via slow intravenous infusion, approximately500 ml of an aqueous solution that is prepared by addition of 5 ml ofthe following stock solution to 500 ml of sterile saline: ComponentConcentration (mg/ml) Urocortin-II  12 Sodium phosphate buffer, pH 7.4140

[0184] As a result of treatment, the subject exhibits measurablepreservation of skeletal muscle mass and function, and reduced physicaltherapy needs during the coma and after regaining consciousness, ascompared to the subject's status without drug therapy.

Example 14

[0185] Therapeutic Treatment of a Patient with Duchenne MuscularDystrophy Using CRF

[0186] A male subject weighing 40 kg with an existing diagnosis ofDuchenne's Muscular Dystrophy is treated with a compound that exhibitsCRF1-R and CRF2-R agonism over a similar dose range. The subject istreated with a sustained-release, depot formulation of the compound inorder to improve or retain muscle strength and function over theprogression of the disease. Specifically, once each month the subject isadministered, via intramuscular injection, 3 ml of an aqueous solutionof pH 6.0 comprising the following: Component Concentration (mg/ml) CRH(Corticotropin-Releasing Hormone) 4 D,L lactic and glycolic acidcopolymer 5

[0187] As a result of the treatment, the subject experiences either animprovement or an attenuation of the decline of muscle strength ormuscle function in timed-function evaluations as compared to thatexhibited during the natural progression of the disease.

[0188] The present invention is not to be limited in scope by thespecific embodiments described which are intended solely asillustrations of individual aspects of the invention, and functionallyequivalent methods and components are within the scope of the invention.These include, but are not limited to, species of test animal, natureand type of CRFR agonists, sex of the animal, model of atrophy, methodof activating CRFR including genetic methodologies, etc. Variousmodifications of the invention, in addition to those shown and describedherein will be apparent to those skilled in the art upon readingforegoing description and accompanying figures. Such modifications areintended to fall within the scope of the appended claims.

1 44 1 2536 DNA homo sapiens CDS (227)..(1474) 1 ggggaaacgg cggccagacttccccgggaa ggggcgagcg agagccgggc cgggccgggc 60 cgggccgcgg ggccgggaagcgccgagccg ggcatctcct caccaggcag cgaccgagga 120 gcccggccgc ccaccccgtgccgcccgagc ccgcagccgc ccgccggtcc ctctgggatg 180 tccgtaggac ccgggcattcaggacggtag ccgagcgagc ccgagg atg gga ggg 235 Met Gly Gly 1 cac ccg cagctc cgt ctc gtc aag gcc ctt ctc ctt ctg ggg ctg aac 283 His Pro Gln LeuArg Leu Val Lys Ala Leu Leu Leu Leu Gly Leu Asn 5 10 15 ccc gtc tct gcctcc ctc cag gac cag cac tgc gag agc ctg tcc ctg 331 Pro Val Ser Ala SerLeu Gln Asp Gln His Cys Glu Ser Leu Ser Leu 20 25 30 35 gcc agc aac atctca gga ctg cag tgc aac gca tcc gtg gac ctc att 379 Ala Ser Asn Ile SerGly Leu Gln Cys Asn Ala Ser Val Asp Leu Ile 40 45 50 ggc acc tgc tgg ccccgc agc cct gcg ggg cag cta gtg gtt cgg ccc 427 Gly Thr Cys Trp Pro ArgSer Pro Ala Gly Gln Leu Val Val Arg Pro 55 60 65 tgc cct gcc ttt ttc tatggt gtc cgc tac aat acc aca aac aat ggc 475 Cys Pro Ala Phe Phe Tyr GlyVal Arg Tyr Asn Thr Thr Asn Asn Gly 70 75 80 tac cgg gag tgc ctg gcc aatggc agc tgg gcc gcc cgc gtg aat tac 523 Tyr Arg Glu Cys Leu Ala Asn GlySer Trp Ala Ala Arg Val Asn Tyr 85 90 95 tcc gag tgc cag gag atc ctc aatgag gag aaa aaa agc aag gtg cac 571 Ser Glu Cys Gln Glu Ile Leu Asn GluGlu Lys Lys Ser Lys Val His 100 105 110 115 tac cat gtc gca gtc atc atcaac tac ctg ggc cac tgt atc tcc ctg 619 Tyr His Val Ala Val Ile Ile AsnTyr Leu Gly His Cys Ile Ser Leu 120 125 130 gtg gcc ctc ctg gtg gcc tttgtc ctc ttt ctg cgg ctc agg agc atc 667 Val Ala Leu Leu Val Ala Phe ValLeu Phe Leu Arg Leu Arg Ser Ile 135 140 145 cgg tgc ctg cga aac atc atccac tgg aac ctc atc tcc gcc ttc atc 715 Arg Cys Leu Arg Asn Ile Ile HisTrp Asn Leu Ile Ser Ala Phe Ile 150 155 160 ctg cgc aac gcc acc tgg ttcgtg gtc cag cta acc atg agc ccc gag 763 Leu Arg Asn Ala Thr Trp Phe ValVal Gln Leu Thr Met Ser Pro Glu 165 170 175 gtc cac cag agc aac gtg ggctgg tgc agg ttg gtg aca gcc gcc tac 811 Val His Gln Ser Asn Val Gly TrpCys Arg Leu Val Thr Ala Ala Tyr 180 185 190 195 aac tac ttc cat gtg accaac ttc ttc tgg atg ttc ggc gag ggc tgc 859 Asn Tyr Phe His Val Thr AsnPhe Phe Trp Met Phe Gly Glu Gly Cys 200 205 210 tac ctg cac aca gcc atcgtg ctc acc tac tcc act gac cgg ctg cgc 907 Tyr Leu His Thr Ala Ile ValLeu Thr Tyr Ser Thr Asp Arg Leu Arg 215 220 225 aaa tgg atg ttc atc tgcatt ggc tgg ggt gtg ccc ttc ccc atc att 955 Lys Trp Met Phe Ile Cys IleGly Trp Gly Val Pro Phe Pro Ile Ile 230 235 240 gtg gcc tgg gcc att gggaag ctg tac tac gac aat gag aag tgc tgg 1003 Val Ala Trp Ala Ile Gly LysLeu Tyr Tyr Asp Asn Glu Lys Cys Trp 245 250 255 ttt ggc aaa agg cct ggggtg tac acc gac tac atc tac cag ggc ccc 1051 Phe Gly Lys Arg Pro Gly ValTyr Thr Asp Tyr Ile Tyr Gln Gly Pro 260 265 270 275 atg atc ctg gtc ctgctg atc aat ttc atc ttc ctt ttc aac atc gtc 1099 Met Ile Leu Val Leu LeuIle Asn Phe Ile Phe Leu Phe Asn Ile Val 280 285 290 cgc atc ctc atg accaag ctc cgg gca tcc acc acg tct gag acc att 1147 Arg Ile Leu Met Thr LysLeu Arg Ala Ser Thr Thr Ser Glu Thr Ile 295 300 305 cag tac agg aag gctgtg aaa gcc act ctg gtg ctg ctg ccc ctc ctg 1195 Gln Tyr Arg Lys Ala ValLys Ala Thr Leu Val Leu Leu Pro Leu Leu 310 315 320 ggc atc acc tac atgctg ttc ttc gtc aat ccc ggg gag gat gag gtc 1243 Gly Ile Thr Tyr Met LeuPhe Phe Val Asn Pro Gly Glu Asp Glu Val 325 330 335 tcc cgg gtc gtc ttcatc tac ttc aac tcc ttc ctg gaa tcc ttc cag 1291 Ser Arg Val Val Phe IleTyr Phe Asn Ser Phe Leu Glu Ser Phe Gln 340 345 350 355 ggc ttc ttt gtgtct gtg ttc tac tgt ttc ctc aat agt gag gtc cgt 1339 Gly Phe Phe Val SerVal Phe Tyr Cys Phe Leu Asn Ser Glu Val Arg 360 365 370 tct gcc atc cggaag agg tgg cac cgg tgg cag gac aag cac tcg atc 1387 Ser Ala Ile Arg LysArg Trp His Arg Trp Gln Asp Lys His Ser Ile 375 380 385 cgt gcc cga gtggcc cgt gcc atg tcc atc ccc acc tcc cca acc cgt 1435 Arg Ala Arg Val AlaArg Ala Met Ser Ile Pro Thr Ser Pro Thr Arg 390 395 400 gtc agc ttt cacagc atc aag cag tcc aca gca gtc tga gctggcaggt 1484 Val Ser Phe His SerIle Lys Gln Ser Thr Ala Val 405 410 415 catggagcag cccccaaaga gctgtggctggggggatgac ggccaggctc cctgaccacc 1544 ctgcctgtgg aggtgacctg ttaggtctcatgcccactcc cccaggagca gctggcactg 1604 acagcctggg ggggccgctc tccccctgcagccgtgcagg actctagctc atgagtggaa 1664 agtcacctac aggactgggc cgggcccagggcctctggct tccctgccca atcctccctg 1724 gagaagggac atgggaatga attgaaatggggcgctggac acctacagca gcacgcatgt 1784 ccctccaagg ctgtcttctc ccagagcacaagaaggccag cccactgggc cctggggctg 1844 ccctcggcaa ccgtggggag gccatttgctgccctggggc atcatgggca actcgtgaca 1904 gcctctgact caccacgatg acgcctctggacctcggtga tgccttccga caccactggg 1964 aaccaagggc cctcactcag gaaccctggagacagaagtc aggtgtcatc atcagacttg 2024 cggccacagc actagagtca cccccccaggcctccagaac cttactggca ctgtggcact 2084 gccaccagca atgccctgcc ttgctgccttcaccctgaac atttagtacc ctgcaggcca 2144 ggccagcttc ccctcactta accaccccataccagtcacc tcctgctcct tttcctcttt 2204 tgtgagaaga tgggggctgg agggggcagagtggcctgtg agcaagagcc aggggtgtcc 2264 cagtcccagc ctctggggca gagcttgtagccctggatgg cctctggggc aggaccacta 2324 gctaagcaag ccaggagaag acccctgcccaagtggctct tgggacaacg tgctgcttac 2384 actccaggtg tggaccggcc gcagcccccactgacctgcc catgtccaga gggactggac 2444 agccagggca gggctttggg gggcactagaagatgagggt gtcggctgtg aggcgggtgg 2504 ctggtataaa taatatttat cttttcaaccag 2536 2 415 PRT homo sapiens 2 Met Gly Gly His Pro Gln Leu Arg Leu ValLys Ala Leu Leu Leu Leu 1 5 10 15 Gly Leu Asn Pro Val Ser Ala Ser LeuGln Asp Gln His Cys Glu Ser 20 25 30 Leu Ser Leu Ala Ser Asn Ile Ser GlyLeu Gln Cys Asn Ala Ser Val 35 40 45 Asp Leu Ile Gly Thr Cys Trp Pro ArgSer Pro Ala Gly Gln Leu Val 50 55 60 Val Arg Pro Cys Pro Ala Phe Phe TyrGly Val Arg Tyr Asn Thr Thr 65 70 75 80 Asn Asn Gly Tyr Arg Glu Cys LeuAla Asn Gly Ser Trp Ala Ala Arg 85 90 95 Val Asn Tyr Ser Glu Cys Gln GluIle Leu Asn Glu Glu Lys Lys Ser 100 105 110 Lys Val His Tyr His Val AlaVal Ile Ile Asn Tyr Leu Gly His Cys 115 120 125 Ile Ser Leu Val Ala LeuLeu Val Ala Phe Val Leu Phe Leu Arg Leu 130 135 140 Arg Ser Ile Arg CysLeu Arg Asn Ile Ile His Trp Asn Leu Ile Ser 145 150 155 160 Ala Phe IleLeu Arg Asn Ala Thr Trp Phe Val Val Gln Leu Thr Met 165 170 175 Ser ProGlu Val His Gln Ser Asn Val Gly Trp Cys Arg Leu Val Thr 180 185 190 AlaAla Tyr Asn Tyr Phe His Val Thr Asn Phe Phe Trp Met Phe Gly 195 200 205Glu Gly Cys Tyr Leu His Thr Ala Ile Val Leu Thr Tyr Ser Thr Asp 210 215220 Arg Leu Arg Lys Trp Met Phe Ile Cys Ile Gly Trp Gly Val Pro Phe 225230 235 240 Pro Ile Ile Val Ala Trp Ala Ile Gly Lys Leu Tyr Tyr Asp AsnGlu 245 250 255 Lys Cys Trp Phe Gly Lys Arg Pro Gly Val Tyr Thr Asp TyrIle Tyr 260 265 270 Gln Gly Pro Met Ile Leu Val Leu Leu Ile Asn Phe IlePhe Leu Phe 275 280 285 Asn Ile Val Arg Ile Leu Met Thr Lys Leu Arg AlaSer Thr Thr Ser 290 295 300 Glu Thr Ile Gln Tyr Arg Lys Ala Val Lys AlaThr Leu Val Leu Leu 305 310 315 320 Pro Leu Leu Gly Ile Thr Tyr Met LeuPhe Phe Val Asn Pro Gly Glu 325 330 335 Asp Glu Val Ser Arg Val Val PheIle Tyr Phe Asn Ser Phe Leu Glu 340 345 350 Ser Phe Gln Gly Phe Phe ValSer Val Phe Tyr Cys Phe Leu Asn Ser 355 360 365 Glu Val Arg Ser Ala IleArg Lys Arg Trp His Arg Trp Gln Asp Lys 370 375 380 His Ser Ile Arg AlaArg Val Ala Arg Ala Met Ser Ile Pro Thr Ser 385 390 395 400 Pro Thr ArgVal Ser Phe His Ser Ile Lys Gln Ser Thr Ala Val 405 410 415 3 1285 DNAHomo sapiens CDS (38)..(1285) 3 cccgggcatt caggacggta gccgagcgag cccgaggatg gga ggg cac ccg cag 55 Met Gly Gly His Pro Gln 1 5 ctc cgt ctc gtcaag gcc ctt ctc ctt ctg ggg ctg aac ccc gtc tct 103 Leu Arg Leu Val LysAla Leu Leu Leu Leu Gly Leu Asn Pro Val Ser 10 15 20 gcc tcc ctc cag gaccag cac tgc gag agc ctg tcc ctg gcc agc aac 151 Ala Ser Leu Gln Asp GlnHis Cys Glu Ser Leu Ser Leu Ala Ser Asn 25 30 35 atc tca gga ctg cag tgcaac gca tcc gtg gac ctc att ggc acc tgc 199 Ile Ser Gly Leu Gln Cys AsnAla Ser Val Asp Leu Ile Gly Thr Cys 40 45 50 tgg ccc cgc agc cct gcg gggcag cta gtg gtt cgg ccc tgc cct gcc 247 Trp Pro Arg Ser Pro Ala Gly GlnLeu Val Val Arg Pro Cys Pro Ala 55 60 65 70 ttt ttc tat ggt gtc cgc tacaat acc aca aac aat ggc tac cgg gag 295 Phe Phe Tyr Gly Val Arg Tyr AsnThr Thr Asn Asn Gly Tyr Arg Glu 75 80 85 tgc ctg gcc aat ggc agc tgg gccgcc cgc gtg aat tac tcc gag tgc 343 Cys Leu Ala Asn Gly Ser Trp Ala AlaArg Val Asn Tyr Ser Glu Cys 90 95 100 cag gag atc ctc aat gag gag aaaaaa agc aag gtg cac tac cat gtc 391 Gln Glu Ile Leu Asn Glu Glu Lys LysSer Lys Val His Tyr His Val 105 110 115 gca gtc atc atc aac tac ctg ggccac tgt atc tcc ctg gtg gcc ctc 439 Ala Val Ile Ile Asn Tyr Leu Gly HisCys Ile Ser Leu Val Ala Leu 120 125 130 ctg gtg gcc ttt gtc ctc ttt ctgcgg ctc agg agc atc cgg tgc ctg 487 Leu Val Ala Phe Val Leu Phe Leu ArgLeu Arg Ser Ile Arg Cys Leu 135 140 145 150 cga aac atc atc cac tgg aacctc atc tcc gcc ttc atc ctg cgc aac 535 Arg Asn Ile Ile His Trp Asn LeuIle Ser Ala Phe Ile Leu Arg Asn 155 160 165 gcc acc tgg ttc gtg gtc cagcta acc atg agc ccc gag gtc cac cag 583 Ala Thr Trp Phe Val Val Gln LeuThr Met Ser Pro Glu Val His Gln 170 175 180 agc aac gtg ggc tgg tgc aggttg gtg aca gcc gcc tac aac tac ttc 631 Ser Asn Val Gly Trp Cys Arg LeuVal Thr Ala Ala Tyr Asn Tyr Phe 185 190 195 cat gtg acc aac ttc ttc tggatg ttc ggc gag ggc tgc tac ctg cac 679 His Val Thr Asn Phe Phe Trp MetPhe Gly Glu Gly Cys Tyr Leu His 200 205 210 aca gcc atc gtg ctc acc tactcc act gac cgg ctg cgc aaa tgg atg 727 Thr Ala Ile Val Leu Thr Tyr SerThr Asp Arg Leu Arg Lys Trp Met 215 220 225 230 ttc atc tgc att ggc tggggt gtg ccc ttc ccc atc att gtg gcc tgg 775 Phe Ile Cys Ile Gly Trp GlyVal Pro Phe Pro Ile Ile Val Ala Trp 235 240 245 gcc att ggg aag ctg tactac gac aat gag aag tgc tgg ttt ggc aaa 823 Ala Ile Gly Lys Leu Tyr TyrAsp Asn Glu Lys Cys Trp Phe Gly Lys 250 255 260 agg cct ggg gtg tac accgac tac atc tac cag ggc ccc atg atc ctg 871 Arg Pro Gly Val Tyr Thr AspTyr Ile Tyr Gln Gly Pro Met Ile Leu 265 270 275 gtc ctg ctg atc aat ttcatc ttc ctt ttc aac atc gtc cgc atc ctc 919 Val Leu Leu Ile Asn Phe IlePhe Leu Phe Asn Ile Val Arg Ile Leu 280 285 290 atg acc aag ctc cgg gcatcc acc acg tct gag acc att cag tac agg 967 Met Thr Lys Leu Arg Ala SerThr Thr Ser Glu Thr Ile Gln Tyr Arg 295 300 305 310 aag gct gtg aaa gccact ctg gtg ctg ctg ccc ctc ctg ggc atc acc 1015 Lys Ala Val Lys Ala ThrLeu Val Leu Leu Pro Leu Leu Gly Ile Thr 315 320 325 tac atg ctg ttc ttcgtc aat ccc ggg gag gat gag gtc tcc cgg gtc 1063 Tyr Met Leu Phe Phe ValAsn Pro Gly Glu Asp Glu Val Ser Arg Val 330 335 340 gtc ttc atc tac ttcaac tcc ttc ctg gaa tcc ttc cag ggc ttc ttt 1111 Val Phe Ile Tyr Phe AsnSer Phe Leu Glu Ser Phe Gln Gly Phe Phe 345 350 355 gtg tct gtg ttc tactgt ttc ctc aat agt gag gtc cgt tct gcc atc 1159 Val Ser Val Phe Tyr CysPhe Leu Asn Ser Glu Val Arg Ser Ala Ile 360 365 370 cgg aag agg tgg caccgg tgg cag gac aag cac tcg atc cgt gcc cga 1207 Arg Lys Arg Trp His ArgTrp Gln Asp Lys His Ser Ile Arg Ala Arg 375 380 385 390 gtg gcc cgt gccatg tcc atc ccc acc tcc cca acc cgt gtc agc ttt 1255 Val Ala Arg Ala MetSer Ile Pro Thr Ser Pro Thr Arg Val Ser Phe 395 400 405 cac agc atc aagcag tcc aca gca gtc tga 1285 His Ser Ile Lys Gln Ser Thr Ala Val 410 4154 415 PRT Homo sapiens 4 Met Gly Gly His Pro Gln Leu Arg Leu Val Lys AlaLeu Leu Leu Leu 1 5 10 15 Gly Leu Asn Pro Val Ser Ala Ser Leu Gln AspGln His Cys Glu Ser 20 25 30 Leu Ser Leu Ala Ser Asn Ile Ser Gly Leu GlnCys Asn Ala Ser Val 35 40 45 Asp Leu Ile Gly Thr Cys Trp Pro Arg Ser ProAla Gly Gln Leu Val 50 55 60 Val Arg Pro Cys Pro Ala Phe Phe Tyr Gly ValArg Tyr Asn Thr Thr 65 70 75 80 Asn Asn Gly Tyr Arg Glu Cys Leu Ala AsnGly Ser Trp Ala Ala Arg 85 90 95 Val Asn Tyr Ser Glu Cys Gln Glu Ile LeuAsn Glu Glu Lys Lys Ser 100 105 110 Lys Val His Tyr His Val Ala Val IleIle Asn Tyr Leu Gly His Cys 115 120 125 Ile Ser Leu Val Ala Leu Leu ValAla Phe Val Leu Phe Leu Arg Leu 130 135 140 Arg Ser Ile Arg Cys Leu ArgAsn Ile Ile His Trp Asn Leu Ile Ser 145 150 155 160 Ala Phe Ile Leu ArgAsn Ala Thr Trp Phe Val Val Gln Leu Thr Met 165 170 175 Ser Pro Glu ValHis Gln Ser Asn Val Gly Trp Cys Arg Leu Val Thr 180 185 190 Ala Ala TyrAsn Tyr Phe His Val Thr Asn Phe Phe Trp Met Phe Gly 195 200 205 Glu GlyCys Tyr Leu His Thr Ala Ile Val Leu Thr Tyr Ser Thr Asp 210 215 220 ArgLeu Arg Lys Trp Met Phe Ile Cys Ile Gly Trp Gly Val Pro Phe 225 230 235240 Pro Ile Ile Val Ala Trp Ala Ile Gly Lys Leu Tyr Tyr Asp Asn Glu 245250 255 Lys Cys Trp Phe Gly Lys Arg Pro Gly Val Tyr Thr Asp Tyr Ile Tyr260 265 270 Gln Gly Pro Met Ile Leu Val Leu Leu Ile Asn Phe Ile Phe LeuPhe 275 280 285 Asn Ile Val Arg Ile Leu Met Thr Lys Leu Arg Ala Ser ThrThr Ser 290 295 300 Glu Thr Ile Gln Tyr Arg Lys Ala Val Lys Ala Thr LeuVal Leu Leu 305 310 315 320 Pro Leu Leu Gly Ile Thr Tyr Met Leu Phe PheVal Asn Pro Gly Glu 325 330 335 Asp Glu Val Ser Arg Val Val Phe Ile TyrPhe Asn Ser Phe Leu Glu 340 345 350 Ser Phe Gln Gly Phe Phe Val Ser ValPhe Tyr Cys Phe Leu Asn Ser 355 360 365 Glu Val Arg Ser Ala Ile Arg LysArg Trp His Arg Trp Gln Asp Lys 370 375 380 His Ser Ile Arg Ala Arg ValAla Arg Ala Met Ser Ile Pro Thr Ser 385 390 395 400 Pro Thr Arg Val SerPhe His Ser Ile Lys Gln Ser Thr Ala Val 405 410 415 5 1146 DNA Homosapiens CDS (19)..(1146) 5 agccgagcga gcccgagg atg gga ggg cac ccg cagctc cgt ctc gtc aag 51 Met Gly Gly His Pro Gln Leu Arg Leu Val Lys 1 510 gcc ctt ctc ctt ctg ggg ctg aac ccc gtc tct gcc tcc ctc cag gac 99Ala Leu Leu Leu Leu Gly Leu Asn Pro Val Ser Ala Ser Leu Gln Asp 15 20 25cag cac tgc gag agc ctg tcc ctg gcc agc aac atc tca gac aat ggc 147 GlnHis Cys Glu Ser Leu Ser Leu Ala Ser Asn Ile Ser Asp Asn Gly 30 35 40 taccgg gag tgc ctg gcc aat ggc agc tgg gcc gcc cgc gtg aat tac 195 Tyr ArgGlu Cys Leu Ala Asn Gly Ser Trp Ala Ala Arg Val Asn Tyr 45 50 55 tcc gagtgc cag gag atc ctc aat gag gag aaa aaa agc aag gtg cac 243 Ser Glu CysGln Glu Ile Leu Asn Glu Glu Lys Lys Ser Lys Val His 60 65 70 75 tac catgtc gca gtc atc atc aac tac ctg ggc cac tgt atc tcc ctg 291 Tyr His ValAla Val Ile Ile Asn Tyr Leu Gly His Cys Ile Ser Leu 80 85 90 gtg gcc ctcctg gtg gcc ttt gtc ctc ttt ctg cgg ctc agg agc atc 339 Val Ala Leu LeuVal Ala Phe Val Leu Phe Leu Arg Leu Arg Ser Ile 95 100 105 cgg tgc ctgcga aac atc atc cac tgg aac ctc atc tcc gcc ttc atc 387 Arg Cys Leu ArgAsn Ile Ile His Trp Asn Leu Ile Ser Ala Phe Ile 110 115 120 ctg cgc aacgcc acc tgg ttc gtg gtc cag cta acc atg agc ccc gag 435 Leu Arg Asn AlaThr Trp Phe Val Val Gln Leu Thr Met Ser Pro Glu 125 130 135 gtc cac cagagc aac gtg ggc tgg tgc agg ttg gtg aca gcc gcc tac 483 Val His Gln SerAsn Val Gly Trp Cys Arg Leu Val Thr Ala Ala Tyr 140 145 150 155 aac tacttc cat gtg acc aac ttc ttc tgg atg ttc ggc gag ggc tgc 531 Asn Tyr PheHis Val Thr Asn Phe Phe Trp Met Phe Gly Glu Gly Cys 160 165 170 tac ctgcac aca gcc atc gtg ctc acc tac tcc act gac cgg ctg cgc 579 Tyr Leu HisThr Ala Ile Val Leu Thr Tyr Ser Thr Asp Arg Leu Arg 175 180 185 aaa tggatg ttc atc tgc att ggc tgg ggt gtg ccc ttc ccc atc att 627 Lys Trp MetPhe Ile Cys Ile Gly Trp Gly Val Pro Phe Pro Ile Ile 190 195 200 gtg gcctgg gcc att ggg aag ctg tac tac gac aat gag aag tgc tgg 675 Val Ala TrpAla Ile Gly Lys Leu Tyr Tyr Asp Asn Glu Lys Cys Trp 205 210 215 ttt ggcaaa agg cct ggg gtg tac acc gac tac atc tac cag ggc ccc 723 Phe Gly LysArg Pro Gly Val Tyr Thr Asp Tyr Ile Tyr Gln Gly Pro 220 225 230 235 atgatc ctg gtc ctg ctg atc aat ttc atc ttc ctt ttc aac atc gtc 771 Met IleLeu Val Leu Leu Ile Asn Phe Ile Phe Leu Phe Asn Ile Val 240 245 250 cgcatc ctc atg acc aag ctc cgg gca tcc acc acg tct gag acc att 819 Arg IleLeu Met Thr Lys Leu Arg Ala Ser Thr Thr Ser Glu Thr Ile 255 260 265 cagtac agg aag gct gtg aaa gcc act ctg gtg ctg ctg ccc ctc ctg 867 Gln TyrArg Lys Ala Val Lys Ala Thr Leu Val Leu Leu Pro Leu Leu 270 275 280 ggcatc acc tac atg ctg ttc ttc gtc aat ccc ggg gag gat gag gtc 915 Gly IleThr Tyr Met Leu Phe Phe Val Asn Pro Gly Glu Asp Glu Val 285 290 295 tcccgg gtc gtc ttc atc tac ttc aac tcc ttc ctg gaa tcc ttc cag 963 Ser ArgVal Val Phe Ile Tyr Phe Asn Ser Phe Leu Glu Ser Phe Gln 300 305 310 315ggc ttc ttt gtg tct gtg ttc tac tgt ttc ctc aat agt gag gtc cgt 1011 GlyPhe Phe Val Ser Val Phe Tyr Cys Phe Leu Asn Ser Glu Val Arg 320 325 330tct gcc atc cgg aag agg tgg cac cgg tgg cag gac aag cac tcg atc 1059 SerAla Ile Arg Lys Arg Trp His Arg Trp Gln Asp Lys His Ser Ile 335 340 345cgt gcc cga gtg gcc cgt gcc atg tcc atc ccc acc tcc cca acc cgt 1107 ArgAla Arg Val Ala Arg Ala Met Ser Ile Pro Thr Ser Pro Thr Arg 350 355 360gtc agc ttt cac agc atc aag cag tcc aca gca gtc tga 1146 Val Ser Phe HisSer Ile Lys Gln Ser Thr Ala Val 365 370 375 6 375 PRT Homo sapiens 6 MetGly Gly His Pro Gln Leu Arg Leu Val Lys Ala Leu Leu Leu Leu 1 5 10 15Gly Leu Asn Pro Val Ser Ala Ser Leu Gln Asp Gln His Cys Glu Ser 20 25 30Leu Ser Leu Ala Ser Asn Ile Ser Asp Asn Gly Tyr Arg Glu Cys Leu 35 40 45Ala Asn Gly Ser Trp Ala Ala Arg Val Asn Tyr Ser Glu Cys Gln Glu 50 55 60Ile Leu Asn Glu Glu Lys Lys Ser Lys Val His Tyr His Val Ala Val 65 70 7580 Ile Ile Asn Tyr Leu Gly His Cys Ile Ser Leu Val Ala Leu Leu Val 85 9095 Ala Phe Val Leu Phe Leu Arg Leu Arg Ser Ile Arg Cys Leu Arg Asn 100105 110 Ile Ile His Trp Asn Leu Ile Ser Ala Phe Ile Leu Arg Asn Ala Thr115 120 125 Trp Phe Val Val Gln Leu Thr Met Ser Pro Glu Val His Gln SerAsn 130 135 140 Val Gly Trp Cys Arg Leu Val Thr Ala Ala Tyr Asn Tyr PheHis Val 145 150 155 160 Thr Asn Phe Phe Trp Met Phe Gly Glu Gly Cys TyrLeu His Thr Ala 165 170 175 Ile Val Leu Thr Tyr Ser Thr Asp Arg Leu ArgLys Trp Met Phe Ile 180 185 190 Cys Ile Gly Trp Gly Val Pro Phe Pro IleIle Val Ala Trp Ala Ile 195 200 205 Gly Lys Leu Tyr Tyr Asp Asn Glu LysCys Trp Phe Gly Lys Arg Pro 210 215 220 Gly Val Tyr Thr Asp Tyr Ile TyrGln Gly Pro Met Ile Leu Val Leu 225 230 235 240 Leu Ile Asn Phe Ile PheLeu Phe Asn Ile Val Arg Ile Leu Met Thr 245 250 255 Lys Leu Arg Ala SerThr Thr Ser Glu Thr Ile Gln Tyr Arg Lys Ala 260 265 270 Val Lys Ala ThrLeu Val Leu Leu Pro Leu Leu Gly Ile Thr Tyr Met 275 280 285 Leu Phe PheVal Asn Pro Gly Glu Asp Glu Val Ser Arg Val Val Phe 290 295 300 Ile TyrPhe Asn Ser Phe Leu Glu Ser Phe Gln Gly Phe Phe Val Ser 305 310 315 320Val Phe Tyr Cys Phe Leu Asn Ser Glu Val Arg Ser Ala Ile Arg Lys 325 330335 Arg Trp His Arg Trp Gln Asp Lys His Ser Ile Arg Ala Arg Val Ala 340345 350 Arg Ala Met Ser Ile Pro Thr Ser Pro Thr Arg Val Ser Phe His Ser355 360 365 Ile Lys Gln Ser Thr Ala Val 370 375 7 1206 DNA Homo sapiensCDS (1)..(1206) 7 atg gga ggg cac ccg cag ctc cgt ctc gtc aag gcc cttctc ctt ctg 48 Met Gly Gly His Pro Gln Leu Arg Leu Val Lys Ala Leu LeuLeu Leu 1 5 10 15 ggg ctg aac ccc gtc tct gcc tcc ctc cag gac cag cactgc gag agc 96 Gly Leu Asn Pro Val Ser Ala Ser Leu Gln Asp Gln His CysGlu Ser 20 25 30 ctg tcc ctg gcc agc aac atc tca gga ctg cag tgc aac gcatcc gtg 144 Leu Ser Leu Ala Ser Asn Ile Ser Gly Leu Gln Cys Asn Ala SerVal 35 40 45 gac ctc att ggc acc tgc tgg ccc cgc agc cct gcg ggg cag ctagtg 192 Asp Leu Ile Gly Thr Cys Trp Pro Arg Ser Pro Ala Gly Gln Leu Val50 55 60 gtt cgg ccc tgc cct gcc ttt ttc tat ggt gtc cgc tac aat acc aca240 Val Arg Pro Cys Pro Ala Phe Phe Tyr Gly Val Arg Tyr Asn Thr Thr 6570 75 80 aac aat ggc tac cgg gag tgc ctg gcc aat ggc agc tgg gcc gcc cgc288 Asn Asn Gly Tyr Arg Glu Cys Leu Ala Asn Gly Ser Trp Ala Ala Arg 8590 95 gtg aat tac tcc gag tgc cag gag atc ctc aat gag gag aaa aaa agc336 Val Asn Tyr Ser Glu Cys Gln Glu Ile Leu Asn Glu Glu Lys Lys Ser 100105 110 aag gtg cac tac cat gtc gca gtc atc atc aac tac ctg ggc cac tgt384 Lys Val His Tyr His Val Ala Val Ile Ile Asn Tyr Leu Gly His Cys 115120 125 atc tcc ctg gtg gcc ctc ctg gtg gcc ttt gtc ctc ttt ctg cgg ctc432 Ile Ser Leu Val Ala Leu Leu Val Ala Phe Val Leu Phe Leu Arg Leu 130135 140 agg agc atc cgg tgc ctg cga aac atc atc cac tgg aac ctc atc tcc480 Arg Ser Ile Arg Cys Leu Arg Asn Ile Ile His Trp Asn Leu Ile Ser 145150 155 160 gcc ttc atc ctg cgc aac gcc acc tgg ttc gtg gtc cag cta accatg 528 Ala Phe Ile Leu Arg Asn Ala Thr Trp Phe Val Val Gln Leu Thr Met165 170 175 agc ccc gag gtc cac cag agc aac gtg ggc tgg tgc agg ttg gtgaca 576 Ser Pro Glu Val His Gln Ser Asn Val Gly Trp Cys Arg Leu Val Thr180 185 190 gcc gcc tac aac tac ttc cat gtg acc aac ttc ttc tgg atg ttcggc 624 Ala Ala Tyr Asn Tyr Phe His Val Thr Asn Phe Phe Trp Met Phe Gly195 200 205 gag ggc tgc tac ctg cac aca gcc atc gtg ctc acc tac tcc actgac 672 Glu Gly Cys Tyr Leu His Thr Ala Ile Val Leu Thr Tyr Ser Thr Asp210 215 220 cgg ctg cgc aaa tgg atg ttc atc tgc att ggc tgg ggt gtg cccttc 720 Arg Leu Arg Lys Trp Met Phe Ile Cys Ile Gly Trp Gly Val Pro Phe225 230 235 240 ccc atc att gtg gcc tgg gcc att ggg aag ctg tac tac gacaat gag 768 Pro Ile Ile Val Ala Trp Ala Ile Gly Lys Leu Tyr Tyr Asp AsnGlu 245 250 255 aag tgc tgg ttt ggc aaa agg cct ggg gtg tac acc gac tacatc tac 816 Lys Cys Trp Phe Gly Lys Arg Pro Gly Val Tyr Thr Asp Tyr IleTyr 260 265 270 cag ggc ccc atg atc ctg gtc ctg ctg atc aat ttc atc ttcctt ttc 864 Gln Gly Pro Met Ile Leu Val Leu Leu Ile Asn Phe Ile Phe LeuPhe 275 280 285 aac atc gtc cgc atc ctc atg acc aag ctc cgg gca tcc accacg tct 912 Asn Ile Val Arg Ile Leu Met Thr Lys Leu Arg Ala Ser Thr ThrSer 290 295 300 gag acc att cag tac agg aag gct gtg aaa gcc act ctg gtgctg ctg 960 Glu Thr Ile Gln Tyr Arg Lys Ala Val Lys Ala Thr Leu Val LeuLeu 305 310 315 320 ccc ctc ctg ggc atc acc tac atg ctg ttc ttc gtc aatccc ggg gag 1008 Pro Leu Leu Gly Ile Thr Tyr Met Leu Phe Phe Val Asn ProGly Glu 325 330 335 gat gag gtc tcc cgg gtc gtc ttc atc tac ttc aac tccttc ctg gaa 1056 Asp Glu Val Ser Arg Val Val Phe Ile Tyr Phe Asn Ser PheLeu Glu 340 345 350 tcc ttc cag gtc cgt tct gcc atc cgg aag agg tgg caccgg tgg cag 1104 Ser Phe Gln Val Arg Ser Ala Ile Arg Lys Arg Trp His ArgTrp Gln 355 360 365 gac aag cac tcg atc cgt gcc cga gtg gcc cgt gcc atgtcc atc ccc 1152 Asp Lys His Ser Ile Arg Ala Arg Val Ala Arg Ala Met SerIle Pro 370 375 380 acc tcc cca acc cgt gtc agc ttt cac agc atc aag cagtcc aca gca 1200 Thr Ser Pro Thr Arg Val Ser Phe His Ser Ile Lys Gln SerThr Ala 385 390 395 400 gtc tga 1206 Val 8 401 PRT Homo sapiens 8 MetGly Gly His Pro Gln Leu Arg Leu Val Lys Ala Leu Leu Leu Leu 1 5 10 15Gly Leu Asn Pro Val Ser Ala Ser Leu Gln Asp Gln His Cys Glu Ser 20 25 30Leu Ser Leu Ala Ser Asn Ile Ser Gly Leu Gln Cys Asn Ala Ser Val 35 40 45Asp Leu Ile Gly Thr Cys Trp Pro Arg Ser Pro Ala Gly Gln Leu Val 50 55 60Val Arg Pro Cys Pro Ala Phe Phe Tyr Gly Val Arg Tyr Asn Thr Thr 65 70 7580 Asn Asn Gly Tyr Arg Glu Cys Leu Ala Asn Gly Ser Trp Ala Ala Arg 85 9095 Val Asn Tyr Ser Glu Cys Gln Glu Ile Leu Asn Glu Glu Lys Lys Ser 100105 110 Lys Val His Tyr His Val Ala Val Ile Ile Asn Tyr Leu Gly His Cys115 120 125 Ile Ser Leu Val Ala Leu Leu Val Ala Phe Val Leu Phe Leu ArgLeu 130 135 140 Arg Ser Ile Arg Cys Leu Arg Asn Ile Ile His Trp Asn LeuIle Ser 145 150 155 160 Ala Phe Ile Leu Arg Asn Ala Thr Trp Phe Val ValGln Leu Thr Met 165 170 175 Ser Pro Glu Val His Gln Ser Asn Val Gly TrpCys Arg Leu Val Thr 180 185 190 Ala Ala Tyr Asn Tyr Phe His Val Thr AsnPhe Phe Trp Met Phe Gly 195 200 205 Glu Gly Cys Tyr Leu His Thr Ala IleVal Leu Thr Tyr Ser Thr Asp 210 215 220 Arg Leu Arg Lys Trp Met Phe IleCys Ile Gly Trp Gly Val Pro Phe 225 230 235 240 Pro Ile Ile Val Ala TrpAla Ile Gly Lys Leu Tyr Tyr Asp Asn Glu 245 250 255 Lys Cys Trp Phe GlyLys Arg Pro Gly Val Tyr Thr Asp Tyr Ile Tyr 260 265 270 Gln Gly Pro MetIle Leu Val Leu Leu Ile Asn Phe Ile Phe Leu Phe 275 280 285 Asn Ile ValArg Ile Leu Met Thr Lys Leu Arg Ala Ser Thr Thr Ser 290 295 300 Glu ThrIle Gln Tyr Arg Lys Ala Val Lys Ala Thr Leu Val Leu Leu 305 310 315 320Pro Leu Leu Gly Ile Thr Tyr Met Leu Phe Phe Val Asn Pro Gly Glu 325 330335 Asp Glu Val Ser Arg Val Val Phe Ile Tyr Phe Asn Ser Phe Leu Glu 340345 350 Ser Phe Gln Val Arg Ser Ala Ile Arg Lys Arg Trp His Arg Trp Gln355 360 365 Asp Lys His Ser Ile Arg Ala Arg Val Ala Arg Ala Met Ser IlePro 370 375 380 Thr Ser Pro Thr Arg Val Ser Phe His Ser Ile Lys Gln SerThr Ala 385 390 395 400 Val 9 2110 DNA Homo sapiens CDS (1)..(1236) 9atg gac gcg gca ctg ctc cac agc ctg ctg gag gcc aac tgc agc ctg 48 MetAsp Ala Ala Leu Leu His Ser Leu Leu Glu Ala Asn Cys Ser Leu 1 5 10 15gcg ctg gct gaa gag ctg ctc ttg gac ggc tgg ggg cca ccc ctg gac 96 AlaLeu Ala Glu Glu Leu Leu Leu Asp Gly Trp Gly Pro Pro Leu Asp 20 25 30 cccgag ggt ccc tac tcc tac tgc aac acg acc ttg gac cag atc gga 144 Pro GluGly Pro Tyr Ser Tyr Cys Asn Thr Thr Leu Asp Gln Ile Gly 35 40 45 acg tgctgg ccc cgc agc gct gcc gga gcc ctc gtg gag agg ccg tgc 192 Thr Cys TrpPro Arg Ser Ala Ala Gly Ala Leu Val Glu Arg Pro Cys 50 55 60 ccc gag tacttc aac ggc gtc aag tac aac acg acc cgg aat gcc tat 240 Pro Glu Tyr PheAsn Gly Val Lys Tyr Asn Thr Thr Arg Asn Ala Tyr 65 70 75 80 cga gaa tgcttg gag aat ggg acg tgg gcc tca aag atc aac tac tca 288 Arg Glu Cys LeuGlu Asn Gly Thr Trp Ala Ser Lys Ile Asn Tyr Ser 85 90 95 cag tgt gag cccatt ttg gat gac aag cag agg aag tat gac ctg cac 336 Gln Cys Glu Pro IleLeu Asp Asp Lys Gln Arg Lys Tyr Asp Leu His 100 105 110 tac cgc atc gccctt gtc gtc aac tac ctg ggc cac tgc gta tct gtg 384 Tyr Arg Ile Ala LeuVal Val Asn Tyr Leu Gly His Cys Val Ser Val 115 120 125 gca gcc ctg gtggcc gcc ttc ctg ctt ttc ctg gcc ctg cgg agc att 432 Ala Ala Leu Val AlaAla Phe Leu Leu Phe Leu Ala Leu Arg Ser Ile 130 135 140 cgc tgt ctg cggaat gtg att cac tgg aac ctc atc acc acc ttt atc 480 Arg Cys Leu Arg AsnVal Ile His Trp Asn Leu Ile Thr Thr Phe Ile 145 150 155 160 ctg cga aatgtc atg tgg ttc ctg ctg cag ctc gtt gac cat gaa gtg 528 Leu Arg Asn ValMet Trp Phe Leu Leu Gln Leu Val Asp His Glu Val 165 170 175 cac gag agcaat gag gtc tgg tgc cac tgc atc acc acc atc ttc aac 576 His Glu Ser AsnGlu Val Trp Cys His Cys Ile Thr Thr Ile Phe Asn 180 185 190 tac ttc gtggtg acc aac ttc ttc tgg atg ttt gtg gaa ggc tgc tac 624 Tyr Phe Val ValThr Asn Phe Phe Trp Met Phe Val Glu Gly Cys Tyr 195 200 205 ctg cac acggcc att gtc atg acc tac tcc act gag cgc ctg cgc aag 672 Leu His Thr AlaIle Val Met Thr Tyr Ser Thr Glu Arg Leu Arg Lys 210 215 220 tgc ctc ttcctc ttc atc gga tgg tgc atc ccc ttc ccc atc atc gtc 720 Cys Leu Phe LeuPhe Ile Gly Trp Cys Ile Pro Phe Pro Ile Ile Val 225 230 235 240 gcc tgggcc atc ggc aag ctc tac tat gag aat gaa cag tgc tgg ttt 768 Ala Trp AlaIle Gly Lys Leu Tyr Tyr Glu Asn Glu Gln Cys Trp Phe 245 250 255 ggc aaggag cct ggc gac ctg gtg gac tac atc tac caa ggc ccc atc 816 Gly Lys GluPro Gly Asp Leu Val Asp Tyr Ile Tyr Gln Gly Pro Ile 260 265 270 att ctcgtg ctc ctg atc aat ttc gta ttt ctg ttc aac atc gtc agg 864 Ile Leu ValLeu Leu Ile Asn Phe Val Phe Leu Phe Asn Ile Val Arg 275 280 285 atc ctaatg aca aag tta cgc gcg tcc acc aca tcc gag aca atc cag 912 Ile Leu MetThr Lys Leu Arg Ala Ser Thr Thr Ser Glu Thr Ile Gln 290 295 300 tac aggaag gca gtg aag gcc acc ctg gtg ctc ctg ccc ctc ctg ggc 960 Tyr Arg LysAla Val Lys Ala Thr Leu Val Leu Leu Pro Leu Leu Gly 305 310 315 320 atcacc tac atg ctc ttc ttc gtc aat ccc ggg gag gac gac ctg tca 1008 Ile ThrTyr Met Leu Phe Phe Val Asn Pro Gly Glu Asp Asp Leu Ser 325 330 335 cagatc atg ttc atc tat ttc aac tcc ttc ctg cag tcg ttc cag ggt 1056 Gln IleMet Phe Ile Tyr Phe Asn Ser Phe Leu Gln Ser Phe Gln Gly 340 345 350 ttcttc gtg tct gtc ttc tac tgc ttc ttc aat gga gag gtg cgc tca 1104 Phe PheVal Ser Val Phe Tyr Cys Phe Phe Asn Gly Glu Val Arg Ser 355 360 365 gccgtg agg aag agg tgg cac cgc tgg cag gac cat cac tcc ctt cga 1152 Ala ValArg Lys Arg Trp His Arg Trp Gln Asp His His Ser Leu Arg 370 375 380 gtcccc atg gcc cgg gcc atg tcc atc cct aca tca ccc aca cgg atc 1200 Val ProMet Ala Arg Ala Met Ser Ile Pro Thr Ser Pro Thr Arg Ile 385 390 395 400agc ttc cac agc atc aag cag acg gcc gct gtg tga cccctcggtc 1246 Ser PheHis Ser Ile Lys Gln Thr Ala Ala Val 405 410 gcccacctgc acagctcccctgtcctcctc caccttcttc ctctgggttc tctgtgctgg 1306 gcaggctctc gtggggcaggagatgggagg ggagagacca gctctccagc ctggcaggaa 1366 agagggggtg cggcagccaagggggactgc aagggacagg gatgagtggg ggccaccagg 1426 ctcagcgcaa gaggaagcagagggaattca caggaccccc tgagaagagc cagtcagatg 1486 tctgcaggca tttgcccatcccagcctctc tggccagggc cttactgggc ccagagcaga 1546 gaaggacctg tccaacacacacagctattt atagtagcag acacagggct cccctgccct 1606 actcatggag ccagcagccaggcaatggtg tggccctgca ctggcccttg gactccacac 1666 tcagtggtgc cctgcagttgggtgggttaa cgccaagcaa aggatcagtt tggctgcctt 1726 atcccagggc tgtcacctagagaggctcac ttgtacccca ccctgttcct gtgtcccctc 1786 cccagccatc ctccccgccttgggggctcc atgaaggatg caggcttcca ggcctggctt 1846 cctctcttgg gagaccccttctctgcctag tccacagatt aggcaatcaa ggaagacgcc 1906 atcagggaag ccacatccttagtcaaccag ttgcatcgtg cggggcaaaa tgaggagcag 1966 aggcatggag gagggaggcgtgggatggga atagcagaac caccatgtct tcagtgattg 2026 aaactcatac cccattgccctttgccctcc agtctcccct tcagaaacat ctctgctctc 2086 tgtgaaataa accatgcctcttgg 2110 10 411 PRT Homo sapiens 10 Met Asp Ala Ala Leu Leu His Ser LeuLeu Glu Ala Asn Cys Ser Leu 1 5 10 15 Ala Leu Ala Glu Glu Leu Leu LeuAsp Gly Trp Gly Pro Pro Leu Asp 20 25 30 Pro Glu Gly Pro Tyr Ser Tyr CysAsn Thr Thr Leu Asp Gln Ile Gly 35 40 45 Thr Cys Trp Pro Arg Ser Ala AlaGly Ala Leu Val Glu Arg Pro Cys 50 55 60 Pro Glu Tyr Phe Asn Gly Val LysTyr Asn Thr Thr Arg Asn Ala Tyr 65 70 75 80 Arg Glu Cys Leu Glu Asn GlyThr Trp Ala Ser Lys Ile Asn Tyr Ser 85 90 95 Gln Cys Glu Pro Ile Leu AspAsp Lys Gln Arg Lys Tyr Asp Leu His 100 105 110 Tyr Arg Ile Ala Leu ValVal Asn Tyr Leu Gly His Cys Val Ser Val 115 120 125 Ala Ala Leu Val AlaAla Phe Leu Leu Phe Leu Ala Leu Arg Ser Ile 130 135 140 Arg Cys Leu ArgAsn Val Ile His Trp Asn Leu Ile Thr Thr Phe Ile 145 150 155 160 Leu ArgAsn Val Met Trp Phe Leu Leu Gln Leu Val Asp His Glu Val 165 170 175 HisGlu Ser Asn Glu Val Trp Cys His Cys Ile Thr Thr Ile Phe Asn 180 185 190Tyr Phe Val Val Thr Asn Phe Phe Trp Met Phe Val Glu Gly Cys Tyr 195 200205 Leu His Thr Ala Ile Val Met Thr Tyr Ser Thr Glu Arg Leu Arg Lys 210215 220 Cys Leu Phe Leu Phe Ile Gly Trp Cys Ile Pro Phe Pro Ile Ile Val225 230 235 240 Ala Trp Ala Ile Gly Lys Leu Tyr Tyr Glu Asn Glu Gln CysTrp Phe 245 250 255 Gly Lys Glu Pro Gly Asp Leu Val Asp Tyr Ile Tyr GlnGly Pro Ile 260 265 270 Ile Leu Val Leu Leu Ile Asn Phe Val Phe Leu PheAsn Ile Val Arg 275 280 285 Ile Leu Met Thr Lys Leu Arg Ala Ser Thr ThrSer Glu Thr Ile Gln 290 295 300 Tyr Arg Lys Ala Val Lys Ala Thr Leu ValLeu Leu Pro Leu Leu Gly 305 310 315 320 Ile Thr Tyr Met Leu Phe Phe ValAsn Pro Gly Glu Asp Asp Leu Ser 325 330 335 Gln Ile Met Phe Ile Tyr PheAsn Ser Phe Leu Gln Ser Phe Gln Gly 340 345 350 Phe Phe Val Ser Val PheTyr Cys Phe Phe Asn Gly Glu Val Arg Ser 355 360 365 Ala Val Arg Lys ArgTrp His Arg Trp Gln Asp His His Ser Leu Arg 370 375 380 Val Pro Met AlaArg Ala Met Ser Ile Pro Thr Ser Pro Thr Arg Ile 385 390 395 400 Ser PheHis Ser Ile Lys Gln Thr Ala Ala Val 405 410 11 1600 DNA Homo sapiens CDS(71)..(1387) 11 ggggctggcc agggtgtgac caccgtgctg ggcagcaggc tccagtccctaacccccagc 60 cactactggc atg agg ggt ccc tca ggg ccc cca ggc ctc ctc tacgtc 109 Met Arg Gly Pro Ser Gly Pro Pro Gly Leu Leu Tyr Val 1 5 10 ccacac ctc ctc ctc tgc ctg ctc tgc ctc ctc cca ccg ccg ctc caa 157 Pro HisLeu Leu Leu Cys Leu Leu Cys Leu Leu Pro Pro Pro Leu Gln 15 20 25 tac gcagcc ggg cag agc cag atg ccc aaa gac cag ccc ctg tgg gca 205 Tyr Ala AlaGly Gln Ser Gln Met Pro Lys Asp Gln Pro Leu Trp Ala 30 35 40 45 ctt ctggag cag tac tgc cac acc atc atg acc ctc acc aac ctc tca 253 Leu Leu GluGln Tyr Cys His Thr Ile Met Thr Leu Thr Asn Leu Ser 50 55 60 ggt ccc tactcc tac tgc aac acg acc ttg gac cag atc gga acg tgc 301 Gly Pro Tyr SerTyr Cys Asn Thr Thr Leu Asp Gln Ile Gly Thr Cys 65 70 75 tgg ccc cgc agcgct gcc gga gcc ctc gtg gag agg ccg tgc ccc gag 349 Trp Pro Arg Ser AlaAla Gly Ala Leu Val Glu Arg Pro Cys Pro Glu 80 85 90 tac ttc aac ggc gtcaag tac aac acg acc cgg aat gcc tat cga gaa 397 Tyr Phe Asn Gly Val LysTyr Asn Thr Thr Arg Asn Ala Tyr Arg Glu 95 100 105 tgc ttg gag aat gggacg tgg gcc tca aag atc aac tac tca cag tgt 445 Cys Leu Glu Asn Gly ThrTrp Ala Ser Lys Ile Asn Tyr Ser Gln Cys 110 115 120 125 gag ccc att ttggat gac aag cag agg aag tat gac ctg cac tac cgc 493 Glu Pro Ile Leu AspAsp Lys Gln Arg Lys Tyr Asp Leu His Tyr Arg 130 135 140 atc gcc ctt gtcgtc aac tac ctg ggc cac tgc gta tct gtg gca gcc 541 Ile Ala Leu Val ValAsn Tyr Leu Gly His Cys Val Ser Val Ala Ala 145 150 155 ctg gtg gcc gccttc ctg ctt ttc ctg gcc ctg cgg agc att cgc tgt 589 Leu Val Ala Ala PheLeu Leu Phe Leu Ala Leu Arg Ser Ile Arg Cys 160 165 170 ctg cgg aat gtgatt cac tgg aac ctc atc acc acc ttt atc ctg cga 637 Leu Arg Asn Val IleHis Trp Asn Leu Ile Thr Thr Phe Ile Leu Arg 175 180 185 aat gtc atg tggttc ctg ctg cag ctc gtt gac cat gaa gtg cac gag 685 Asn Val Met Trp PheLeu Leu Gln Leu Val Asp His Glu Val His Glu 190 195 200 205 agc aat gaggtc tgg tgc cgc tgc atc acc acc atc ttc aac tac ttc 733 Ser Asn Glu ValTrp Cys Arg Cys Ile Thr Thr Ile Phe Asn Tyr Phe 210 215 220 gtg gtg accaac ttc ttc tgg atg ttt gtg gaa ggc tgc tac ctg cac 781 Val Val Thr AsnPhe Phe Trp Met Phe Val Glu Gly Cys Tyr Leu His 225 230 235 acg gcc attgtc atg acc tac tcc act gag cgc ctg cgc aag tgc ctc 829 Thr Ala Ile ValMet Thr Tyr Ser Thr Glu Arg Leu Arg Lys Cys Leu 240 245 250 ttc ctc ttcatc gga tgg tgc atc ccc ttc ccc atc atc gtc gcc tgg 877 Phe Leu Phe IleGly Trp Cys Ile Pro Phe Pro Ile Ile Val Ala Trp 255 260 265 gcc atc ggcaag ctc tac tat gag aat gaa cag tgc tgg ttt ggc aag 925 Ala Ile Gly LysLeu Tyr Tyr Glu Asn Glu Gln Cys Trp Phe Gly Lys 270 275 280 285 gag cctggc gac ctg gtg gac tac atc tac caa ggc ccc atc att ctc 973 Glu Pro GlyAsp Leu Val Asp Tyr Ile Tyr Gln Gly Pro Ile Ile Leu 290 295 300 gtg ctcctg atc aat ttc gta ttt ctg ttc aac atc gtc agg atc cta 1021 Val Leu LeuIle Asn Phe Val Phe Leu Phe Asn Ile Val Arg Ile Leu 305 310 315 atg acaaag tta cgc gcg tcc acc aca tcc gag aca atc cag tac agg 1069 Met Thr LysLeu Arg Ala Ser Thr Thr Ser Glu Thr Ile Gln Tyr Arg 320 325 330 aag gcagtg aag gcc acc ctg gtg ctc ctg ccc ctc ctg ggc atc acc 1117 Lys Ala ValLys Ala Thr Leu Val Leu Leu Pro Leu Leu Gly Ile Thr 335 340 345 tac atgctc ttc ttc gtc aat ccc ggg gag gac gac ctg tca cag atc 1165 Tyr Met LeuPhe Phe Val Asn Pro Gly Glu Asp Asp Leu Ser Gln Ile 350 355 360 365 atgttc atc tat ttc aac tcc ttc ctg cag tcg ttc cag ggt ttc ttc 1213 Met PheIle Tyr Phe Asn Ser Phe Leu Gln Ser Phe Gln Gly Phe Phe 370 375 380 gtgtct gtc ttc tac tgc ttc ttc aat gga gag gtg cgc tca gcc gtg 1261 Val SerVal Phe Tyr Cys Phe Phe Asn Gly Glu Val Arg Ser Ala Val 385 390 395 aggaag agg tgg cac cgc tgg cag gac cat cac tcc ctt cga gtc ccc 1309 Arg LysArg Trp His Arg Trp Gln Asp His His Ser Leu Arg Val Pro 400 405 410 atggcc cgg gcc atg tcc atc cct aca tca ccc aca cgg atc agc ttc 1357 Met AlaArg Ala Met Ser Ile Pro Thr Ser Pro Thr Arg Ile Ser Phe 415 420 425 cacagc atc aag cag acg gcc gct gtg tga cccctcggtc gcccacctgc 1407 His SerIle Lys Gln Thr Ala Ala Val 430 435 acagctcccc tgtcctcctc caccttcttcctctgggttc tctgtgctgg gcaggctctc 1467 gtggggcagg agatgggagg ggagagaccagctctccagc ctggcaggaa agagggggtg 1527 cggcagccaa gggggactgc aagggacagggatgagtggg ggccaccagg ctcagcgcaa 1587 gaggaagcag agg 1600 12 438 PRTHomo sapiens 12 Met Arg Gly Pro Ser Gly Pro Pro Gly Leu Leu Tyr Val ProHis Leu 1 5 10 15 Leu Leu Cys Leu Leu Cys Leu Leu Pro Pro Pro Leu GlnTyr Ala Ala 20 25 30 Gly Gln Ser Gln Met Pro Lys Asp Gln Pro Leu Trp AlaLeu Leu Glu 35 40 45 Gln Tyr Cys His Thr Ile Met Thr Leu Thr Asn Leu SerGly Pro Tyr 50 55 60 Ser Tyr Cys Asn Thr Thr Leu Asp Gln Ile Gly Thr CysTrp Pro Arg 65 70 75 80 Ser Ala Ala Gly Ala Leu Val Glu Arg Pro Cys ProGlu Tyr Phe Asn 85 90 95 Gly Val Lys Tyr Asn Thr Thr Arg Asn Ala Tyr ArgGlu Cys Leu Glu 100 105 110 Asn Gly Thr Trp Ala Ser Lys Ile Asn Tyr SerGln Cys Glu Pro Ile 115 120 125 Leu Asp Asp Lys Gln Arg Lys Tyr Asp LeuHis Tyr Arg Ile Ala Leu 130 135 140 Val Val Asn Tyr Leu Gly His Cys ValSer Val Ala Ala Leu Val Ala 145 150 155 160 Ala Phe Leu Leu Phe Leu AlaLeu Arg Ser Ile Arg Cys Leu Arg Asn 165 170 175 Val Ile His Trp Asn LeuIle Thr Thr Phe Ile Leu Arg Asn Val Met 180 185 190 Trp Phe Leu Leu GlnLeu Val Asp His Glu Val His Glu Ser Asn Glu 195 200 205 Val Trp Cys ArgCys Ile Thr Thr Ile Phe Asn Tyr Phe Val Val Thr 210 215 220 Asn Phe PheTrp Met Phe Val Glu Gly Cys Tyr Leu His Thr Ala Ile 225 230 235 240 ValMet Thr Tyr Ser Thr Glu Arg Leu Arg Lys Cys Leu Phe Leu Phe 245 250 255Ile Gly Trp Cys Ile Pro Phe Pro Ile Ile Val Ala Trp Ala Ile Gly 260 265270 Lys Leu Tyr Tyr Glu Asn Glu Gln Cys Trp Phe Gly Lys Glu Pro Gly 275280 285 Asp Leu Val Asp Tyr Ile Tyr Gln Gly Pro Ile Ile Leu Val Leu Leu290 295 300 Ile Asn Phe Val Phe Leu Phe Asn Ile Val Arg Ile Leu Met ThrLys 305 310 315 320 Leu Arg Ala Ser Thr Thr Ser Glu Thr Ile Gln Tyr ArgLys Ala Val 325 330 335 Lys Ala Thr Leu Val Leu Leu Pro Leu Leu Gly IleThr Tyr Met Leu 340 345 350 Phe Phe Val Asn Pro Gly Glu Asp Asp Leu SerGln Ile Met Phe Ile 355 360 365 Tyr Phe Asn Ser Phe Leu Gln Ser Phe GlnGly Phe Phe Val Ser Val 370 375 380 Phe Tyr Cys Phe Phe Asn Gly Glu ValArg Ser Ala Val Arg Lys Arg 385 390 395 400 Trp His Arg Trp Gln Asp HisHis Ser Leu Arg Val Pro Met Ala Arg 405 410 415 Ala Met Ser Ile Pro ThrSer Pro Thr Arg Ile Ser Phe His Ser Ile 420 425 430 Lys Gln Thr Ala AlaVal 435 13 1558 DNA Homo sapiens CDS (152)..(1345) 13 ctgtgctcaagcaatctgcc taccttggct tccccaagtg ctgagattat gggtgtgagc 60 cactgcacctggccaagaat ccgaatggat tcaaagatac cttgaaataa ttcctcaatg 120 caacacacacacatatgcca gggttggtca a atg gga aga gag cct tgg cct 172 Met Gly Arg GluPro Trp Pro 1 5 gaa gac agg gac ctg ggc ttt cct cag ctc ttc tgc caa ggtccc tac 220 Glu Asp Arg Asp Leu Gly Phe Pro Gln Leu Phe Cys Gln Gly ProTyr 10 15 20 tcc tac tgc aac acg acc ttg gac cag atc gga acg tgc tgg ccccgc 268 Ser Tyr Cys Asn Thr Thr Leu Asp Gln Ile Gly Thr Cys Trp Pro Arg25 30 35 agc gct gcc gga gcc ctc gtg gag agg ccg tgc ccc gag tac ttc aac316 Ser Ala Ala Gly Ala Leu Val Glu Arg Pro Cys Pro Glu Tyr Phe Asn 4045 50 55 ggc gtc aag tac aac acg acc cgg aat gcc tat cga gaa tgc ttg gag364 Gly Val Lys Tyr Asn Thr Thr Arg Asn Ala Tyr Arg Glu Cys Leu Glu 6065 70 aat ggg acg tgg gcc tca aag atc aac tac tca cag tgt gag ccc att412 Asn Gly Thr Trp Ala Ser Lys Ile Asn Tyr Ser Gln Cys Glu Pro Ile 7580 85 ttg gat gac aag cag agg aag tat gac ctg cac tac cgc atc gcc ctt460 Leu Asp Asp Lys Gln Arg Lys Tyr Asp Leu His Tyr Arg Ile Ala Leu 9095 100 gtc gtc aac tac ctg ggc cac tgc gta tct gtg gca gcc ctg gtg gcc508 Val Val Asn Tyr Leu Gly His Cys Val Ser Val Ala Ala Leu Val Ala 105110 115 gcc ttc ctg ctt ttc ctg gcc ctg cgg agc att cgc tgt ctg cgg aat556 Ala Phe Leu Leu Phe Leu Ala Leu Arg Ser Ile Arg Cys Leu Arg Asn 120125 130 135 gtg att cac tgg aac ctc atc acc acc ttt atc ctg cga aat gtcatg 604 Val Ile His Trp Asn Leu Ile Thr Thr Phe Ile Leu Arg Asn Val Met140 145 150 tgg ttc ctg ctg cag ctc gtt gac cat gaa gtg cac gag agc aatgag 652 Trp Phe Leu Leu Gln Leu Val Asp His Glu Val His Glu Ser Asn Glu155 160 165 gtc tgg tgc cgc tgc atc acc acc atc ttc aac tac ttc gtg gtgacc 700 Val Trp Cys Arg Cys Ile Thr Thr Ile Phe Asn Tyr Phe Val Val Thr170 175 180 aac ttc ttc tgg atg ttt gtg gaa ggc tgc tac ctg cac acg gccatt 748 Asn Phe Phe Trp Met Phe Val Glu Gly Cys Tyr Leu His Thr Ala Ile185 190 195 gtc atg acc tac tcc act gag cgc ctg cgc aag tgc ctc ttc ctcttc 796 Val Met Thr Tyr Ser Thr Glu Arg Leu Arg Lys Cys Leu Phe Leu Phe200 205 210 215 atc gga tgg tgc atc ccc ttc ccc atc atc gtc gcc tgg gccatc ggc 844 Ile Gly Trp Cys Ile Pro Phe Pro Ile Ile Val Ala Trp Ala IleGly 220 225 230 aag ctc tac tat gag aat gaa cag tgc tgg ttt ggc aag gagcct ggc 892 Lys Leu Tyr Tyr Glu Asn Glu Gln Cys Trp Phe Gly Lys Glu ProGly 235 240 245 gac ctg gtg gac tac atc tac caa ggc ccc atc att ctc gtgctc ctg 940 Asp Leu Val Asp Tyr Ile Tyr Gln Gly Pro Ile Ile Leu Val LeuLeu 250 255 260 atc aat ttc gta ttt ctg ttc aac atc gtc agg atc cta atgaca aag 988 Ile Asn Phe Val Phe Leu Phe Asn Ile Val Arg Ile Leu Met ThrLys 265 270 275 tta cgc gcg tcc acc aca tcc gag aca atc cag tac agg aaggca gtg 1036 Leu Arg Ala Ser Thr Thr Ser Glu Thr Ile Gln Tyr Arg Lys AlaVal 280 285 290 295 aag gcc acc ctg gtg ctc ctg ccc ctc ctg ggc atc acctac atg ctc 1084 Lys Ala Thr Leu Val Leu Leu Pro Leu Leu Gly Ile Thr TyrMet Leu 300 305 310 ttc ttc gtc aat ccc ggg gag gac gac ctg tca cag atcatg ttc atc 1132 Phe Phe Val Asn Pro Gly Glu Asp Asp Leu Ser Gln Ile MetPhe Ile 315 320 325 tat ttc aac tcc ttc ctg cag tcg ttc cag ggt ttc ttcgtg tct gtc 1180 Tyr Phe Asn Ser Phe Leu Gln Ser Phe Gln Gly Phe Phe ValSer Val 330 335 340 ttc tac tgc ttc ttc aat gga gag gtg cgc tca gcc gtgagg aag agg 1228 Phe Tyr Cys Phe Phe Asn Gly Glu Val Arg Ser Ala Val ArgLys Arg 345 350 355 tgg cac cgc tgg cag gac cat cac tcc ctt cga gtc cccatg gcc cgg 1276 Trp His Arg Trp Gln Asp His His Ser Leu Arg Val Pro MetAla Arg 360 365 370 375 gcc atg tcc atc cct aca tca ccc aca cgg atc agcttc cac agc atc 1324 Ala Met Ser Ile Pro Thr Ser Pro Thr Arg Ile Ser PheHis Ser Ile 380 385 390 aag cag acg gcc gct gtg tga cccctcggtcgcccacctgc acagctcccc 1375 Lys Gln Thr Ala Ala Val 395 tgtcctcctccaccttcttc ctctgggttc tctgtgctgg gcaggctctc gtggggcagg 1435 agatgggaggggagagacca gctctccagc ctggcaggaa agagggggtg cggcagccaa 1495 gggggactgcaagggacagg gatgagtggg ggccaccagg ctcagcgcaa gaggaagcag 1555 agg 1558 14397 PRT Homo sapiens 14 Met Gly Arg Glu Pro Trp Pro Glu Asp Arg Asp LeuGly Phe Pro Gln 1 5 10 15 Leu Phe Cys Gln Gly Pro Tyr Ser Tyr Cys AsnThr Thr Leu Asp Gln 20 25 30 Ile Gly Thr Cys Trp Pro Arg Ser Ala Ala GlyAla Leu Val Glu Arg 35 40 45 Pro Cys Pro Glu Tyr Phe Asn Gly Val Lys TyrAsn Thr Thr Arg Asn 50 55 60 Ala Tyr Arg Glu Cys Leu Glu Asn Gly Thr TrpAla Ser Lys Ile Asn 65 70 75 80 Tyr Ser Gln Cys Glu Pro Ile Leu Asp AspLys Gln Arg Lys Tyr Asp 85 90 95 Leu His Tyr Arg Ile Ala Leu Val Val AsnTyr Leu Gly His Cys Val 100 105 110 Ser Val Ala Ala Leu Val Ala Ala PheLeu Leu Phe Leu Ala Leu Arg 115 120 125 Ser Ile Arg Cys Leu Arg Asn ValIle His Trp Asn Leu Ile Thr Thr 130 135 140 Phe Ile Leu Arg Asn Val MetTrp Phe Leu Leu Gln Leu Val Asp His 145 150 155 160 Glu Val His Glu SerAsn Glu Val Trp Cys Arg Cys Ile Thr Thr Ile 165 170 175 Phe Asn Tyr PheVal Val Thr Asn Phe Phe Trp Met Phe Val Glu Gly 180 185 190 Cys Tyr LeuHis Thr Ala Ile Val Met Thr Tyr Ser Thr Glu Arg Leu 195 200 205 Arg LysCys Leu Phe Leu Phe Ile Gly Trp Cys Ile Pro Phe Pro Ile 210 215 220 IleVal Ala Trp Ala Ile Gly Lys Leu Tyr Tyr Glu Asn Glu Gln Cys 225 230 235240 Trp Phe Gly Lys Glu Pro Gly Asp Leu Val Asp Tyr Ile Tyr Gln Gly 245250 255 Pro Ile Ile Leu Val Leu Leu Ile Asn Phe Val Phe Leu Phe Asn Ile260 265 270 Val Arg Ile Leu Met Thr Lys Leu Arg Ala Ser Thr Thr Ser GluThr 275 280 285 Ile Gln Tyr Arg Lys Ala Val Lys Ala Thr Leu Val Leu LeuPro Leu 290 295 300 Leu Gly Ile Thr Tyr Met Leu Phe Phe Val Asn Pro GlyGlu Asp Asp 305 310 315 320 Leu Ser Gln Ile Met Phe Ile Tyr Phe Asn SerPhe Leu Gln Ser Phe 325 330 335 Gln Gly Phe Phe Val Ser Val Phe Tyr CysPhe Phe Asn Gly Glu Val 340 345 350 Arg Ser Ala Val Arg Lys Arg Trp HisArg Trp Gln Asp His His Ser 355 360 365 Leu Arg Val Pro Met Ala Arg AlaMet Ser Ile Pro Thr Ser Pro Thr 370 375 380 Arg Ile Ser Phe His Ser IleLys Gln Thr Ala Ala Val 385 390 395 15 1411 DNA Rattus norvegicus CDS(80)..(1327) 15 agaccgcagc cgcccgccct ccgctctggg atgtcggagc gatccaggcatccaggacgc 60 tgacggagcg agcccgagg atg gga cgg cgc ccg cag ctc cgg ctcgtg aag 112 Met Gly Arg Arg Pro Gln Leu Arg Leu Val Lys 1 5 10 gcc cttctc ctt ctg ggg ctg aac cct gtg tcc acc tcc ctt cag gat 160 Ala Leu LeuLeu Leu Gly Leu Asn Pro Val Ser Thr Ser Leu Gln Asp 15 20 25 cag cgc tgtgag aac ctg tcc ctg acc agc aat gtt tct ggc ctg cag 208 Gln Arg Cys GluAsn Leu Ser Leu Thr Ser Asn Val Ser Gly Leu Gln 30 35 40 tgc aat gca tccgtg gac ctc att ggc acc tgc tgg ccc cgg agc cct 256 Cys Asn Ala Ser ValAsp Leu Ile Gly Thr Cys Trp Pro Arg Ser Pro 45 50 55 gcg ggc cag ttg gtggtc cga ccc tgc cct gcc ttt ttc tac ggt gtc 304 Ala Gly Gln Leu Val ValArg Pro Cys Pro Ala Phe Phe Tyr Gly Val 60 65 70 75 cgc tac aac acg acaaac aat ggc tac cgg gag tgc ctg gcc aac ggc 352 Arg Tyr Asn Thr Thr AsnAsn Gly Tyr Arg Glu Cys Leu Ala Asn Gly 80 85 90 agc tgg gca gcc cgt gtgaat tat tct gag tgc cag gag att ctc aac 400 Ser Trp Ala Ala Arg Val AsnTyr Ser Glu Cys Gln Glu Ile Leu Asn 95 100 105 gaa gag aag aag agc aaagta cac tac cat gtt gca gtc atc atc aac 448 Glu Glu Lys Lys Ser Lys ValHis Tyr His Val Ala Val Ile Ile Asn 110 115 120 tac ctg ggt cac tgc atctcc ctg gta gcc ctc ctg gtg gcc ttt gtc 496 Tyr Leu Gly His Cys Ile SerLeu Val Ala Leu Leu Val Ala Phe Val 125 130 135 ctc ttc ttg cgg ctc aggagc atc cgg tgc ctg aga aac atc atc cac 544 Leu Phe Leu Arg Leu Arg SerIle Arg Cys Leu Arg Asn Ile Ile His 140 145 150 155 tgg aac ctc atc tcggct ttc atc cta cgc aac gcc acg tgg ttt gtg 592 Trp Asn Leu Ile Ser AlaPhe Ile Leu Arg Asn Ala Thr Trp Phe Val 160 165 170 gtc cag ctc acc gtgagc ccc gag gtg cac cag agc aat gtg gcc tgg 640 Val Gln Leu Thr Val SerPro Glu Val His Gln Ser Asn Val Ala Trp 175 180 185 tgt agg ttg gtg acagcc gcc tac aat tac ttc cat gta acc aac ttc 688 Cys Arg Leu Val Thr AlaAla Tyr Asn Tyr Phe His Val Thr Asn Phe 190 195 200 ttc tgg atg ttc ggtgag ggc tgc tac ctg cac aca gcc att gtg ctc 736 Phe Trp Met Phe Gly GluGly Cys Tyr Leu His Thr Ala Ile Val Leu 205 210 215 acg tac tcc acc gaccgt ctg cgc aag tgg atg ttc gtc tgc att ggc 784 Thr Tyr Ser Thr Asp ArgLeu Arg Lys Trp Met Phe Val Cys Ile Gly 220 225 230 235 tgg ggt gta cctttc ccc atc att gtg gct tgg gcc att ggg aag ctg 832 Trp Gly Val Pro PhePro Ile Ile Val Ala Trp Ala Ile Gly Lys Leu 240 245 250 cac tac gac aatgaa aag tgc tgg ttt ggc aaa cgt cct ggg gta tac 880 His Tyr Asp Asn GluLys Cys Trp Phe Gly Lys Arg Pro Gly Val Tyr 255 260 265 act gac tac atctac cag ggc ccc atg atc ctg gtc ctg ctg atc aac 928 Thr Asp Tyr Ile TyrGln Gly Pro Met Ile Leu Val Leu Leu Ile Asn 270 275 280 ttt atc ttt ctcttc aac att gtc cgc atc ctc atg acc aaa ctc cgg 976 Phe Ile Phe Leu PheAsn Ile Val Arg Ile Leu Met Thr Lys Leu Arg 285 290 295 gca tcc act acatct gag acc att cag tac agg aag gct gtg aag gcc 1024 Ala Ser Thr Thr SerGlu Thr Ile Gln Tyr Arg Lys Ala Val Lys Ala 300 305 310 315 act ctg gtgctc ctg ccc ctt ctg ggc atc acc tac atg ttg ttc ttc 1072 Thr Leu Val LeuLeu Pro Leu Leu Gly Ile Thr Tyr Met Leu Phe Phe 320 325 330 gtc aac cctgga gag gac gag gtc tcc agg gtc gtc ttc atc tac ttc 1120 Val Asn Pro GlyGlu Asp Glu Val Ser Arg Val Val Phe Ile Tyr Phe 335 340 345 aac tct tttctg gag tcc ttt cag ggc ttc ttt gtg tct gtg ttc tac 1168 Asn Ser Phe LeuGlu Ser Phe Gln Gly Phe Phe Val Ser Val Phe Tyr 350 355 360 tgt ttt ctgaac agt gag gtc cgc tcc gct atc cgg aag agg tgg cgt 1216 Cys Phe Leu AsnSer Glu Val Arg Ser Ala Ile Arg Lys Arg Trp Arg 365 370 375 cgg tgg caggac aag cac tcc atc aga gcc cga gtg gcc cga gct atg 1264 Arg Trp Gln AspLys His Ser Ile Arg Ala Arg Val Ala Arg Ala Met 380 385 390 395 tcc atcccc acc tcc ccg acc aga gtc agc ttt cac agc atc aag cag 1312 Ser Ile ProThr Ser Pro Thr Arg Val Ser Phe His Ser Ile Lys Gln 400 405 410 tcc acagca gtg tga gctccaggcc acagagcagc ccccaagacc tgaggccggg 1367 Ser Thr AlaVal 415 gagatgatgc aagctcactg acgagccagt ctgcagacgc aagc 1411 16 415 PRTRattus norvegicus 16 Met Gly Arg Arg Pro Gln Leu Arg Leu Val Lys Ala LeuLeu Leu Leu 1 5 10 15 Gly Leu Asn Pro Val Ser Thr Ser Leu Gln Asp GlnArg Cys Glu Asn 20 25 30 Leu Ser Leu Thr Ser Asn Val Ser Gly Leu Gln CysAsn Ala Ser Val 35 40 45 Asp Leu Ile Gly Thr Cys Trp Pro Arg Ser Pro AlaGly Gln Leu Val 50 55 60 Val Arg Pro Cys Pro Ala Phe Phe Tyr Gly Val ArgTyr Asn Thr Thr 65 70 75 80 Asn Asn Gly Tyr Arg Glu Cys Leu Ala Asn GlySer Trp Ala Ala Arg 85 90 95 Val Asn Tyr Ser Glu Cys Gln Glu Ile Leu AsnGlu Glu Lys Lys Ser 100 105 110 Lys Val His Tyr His Val Ala Val Ile IleAsn Tyr Leu Gly His Cys 115 120 125 Ile Ser Leu Val Ala Leu Leu Val AlaPhe Val Leu Phe Leu Arg Leu 130 135 140 Arg Ser Ile Arg Cys Leu Arg AsnIle Ile His Trp Asn Leu Ile Ser 145 150 155 160 Ala Phe Ile Leu Arg AsnAla Thr Trp Phe Val Val Gln Leu Thr Val 165 170 175 Ser Pro Glu Val HisGln Ser Asn Val Ala Trp Cys Arg Leu Val Thr 180 185 190 Ala Ala Tyr AsnTyr Phe His Val Thr Asn Phe Phe Trp Met Phe Gly 195 200 205 Glu Gly CysTyr Leu His Thr Ala Ile Val Leu Thr Tyr Ser Thr Asp 210 215 220 Arg LeuArg Lys Trp Met Phe Val Cys Ile Gly Trp Gly Val Pro Phe 225 230 235 240Pro Ile Ile Val Ala Trp Ala Ile Gly Lys Leu His Tyr Asp Asn Glu 245 250255 Lys Cys Trp Phe Gly Lys Arg Pro Gly Val Tyr Thr Asp Tyr Ile Tyr 260265 270 Gln Gly Pro Met Ile Leu Val Leu Leu Ile Asn Phe Ile Phe Leu Phe275 280 285 Asn Ile Val Arg Ile Leu Met Thr Lys Leu Arg Ala Ser Thr ThrSer 290 295 300 Glu Thr Ile Gln Tyr Arg Lys Ala Val Lys Ala Thr Leu ValLeu Leu 305 310 315 320 Pro Leu Leu Gly Ile Thr Tyr Met Leu Phe Phe ValAsn Pro Gly Glu 325 330 335 Asp Glu Val Ser Arg Val Val Phe Ile Tyr PheAsn Ser Phe Leu Glu 340 345 350 Ser Phe Gln Gly Phe Phe Val Ser Val PheTyr Cys Phe Leu Asn Ser 355 360 365 Glu Val Arg Ser Ala Ile Arg Lys ArgTrp Arg Arg Trp Gln Asp Lys 370 375 380 His Ser Ile Arg Ala Arg Val AlaArg Ala Met Ser Ile Pro Thr Ser 385 390 395 400 Pro Thr Arg Val Ser PheHis Ser Ile Lys Gln Ser Thr Ala Val 405 410 415 17 1626 DNA Rattusnorvegicus CDS (216)..(1451) 17 gcggcccctc atctccgtga gccccgaggcttctcttggc caaggtccta ggagtgatcc 60 gattgagagc ggcgccccaa agctgccgggctggccgggg tgggcgggga ggcacctgga 120 cgctgcactc tctggtggct ccgcgtcgcgccaggtccct cgcagccacg cggggcgcgc 180 actcccactc ccaacgcgcg cggctccggagcgca atg gac gcg gcg ctg ctc 233 Met Asp Ala Ala Leu Leu 1 5 ctc agcctg ctg gag gcc aac tgc agc ctg gca ctg gcc gaa gag ctg 281 Leu Ser LeuLeu Glu Ala Asn Cys Ser Leu Ala Leu Ala Glu Glu Leu 10 15 20 ctt ttg gacggc tgg gga gag ccc ccg gac ccc gaa ggt ccc tac tcc 329 Leu Leu Asp GlyTrp Gly Glu Pro Pro Asp Pro Glu Gly Pro Tyr Ser 25 30 35 tac tgc aac acgacc ttg gac cag atc ggg acc tgc tgg ccc cag agc 377 Tyr Cys Asn Thr ThrLeu Asp Gln Ile Gly Thr Cys Trp Pro Gln Ser 40 45 50 gcg cct gga gcc ctagtg gag aga cca tgc ccc gaa tac ttc aac ggc 425 Ala Pro Gly Ala Leu ValGlu Arg Pro Cys Pro Glu Tyr Phe Asn Gly 55 60 65 70 atc aag tac aac acgacc cgg aat gcc tac aga gaa tgc ctg gag aat 473 Ile Lys Tyr Asn Thr ThrArg Asn Ala Tyr Arg Glu Cys Leu Glu Asn 75 80 85 ggg acc tgg gcc tca aggatc aac tac tca cac tgt gaa ccc att ttg 521 Gly Thr Trp Ala Ser Arg IleAsn Tyr Ser His Cys Glu Pro Ile Leu 90 95 100 gat gac aag cag agg aagtat gac ctg cat tac cga atc gcc ctc atc 569 Asp Asp Lys Gln Arg Lys TyrAsp Leu His Tyr Arg Ile Ala Leu Ile 105 110 115 atc aac tac ctg ggc cactgt gtt tcc gtg gtg gcc ctg gtg gct gct 617 Ile Asn Tyr Leu Gly His CysVal Ser Val Val Ala Leu Val Ala Ala 120 125 130 ttc ctg ctt ttc cta gtgctg cgg agt atc cgc tgc ctg cgg aat gtg 665 Phe Leu Leu Phe Leu Val LeuArg Ser Ile Arg Cys Leu Arg Asn Val 135 140 145 150 atc cac tgg aac ctcatc acc acc ttc atc ctg aga aac atc acg tgg 713 Ile His Trp Asn Leu IleThr Thr Phe Ile Leu Arg Asn Ile Thr Trp 155 160 165 ttc ctg ctg caa ctcatc gac cac gaa gtg cat gag ggc aat gag gtc 761 Phe Leu Leu Gln Leu IleAsp His Glu Val His Glu Gly Asn Glu Val 170 175 180 tgg tgc cgc tgc gtcacc acc ata ttc aac tac ttt gtg gtc acc aac 809 Trp Cys Arg Cys Val ThrThr Ile Phe Asn Tyr Phe Val Val Thr Asn 185 190 195 ttc ttc tgg atg tttgtg gaa ggc tgc tac ctg cac acg gcc atc gtc 857 Phe Phe Trp Met Phe ValGlu Gly Cys Tyr Leu His Thr Ala Ile Val 200 205 210 atg acg tac tcc acggag cat ctg cgc aag tgg ctc ttc ctc ttc att 905 Met Thr Tyr Ser Thr GluHis Leu Arg Lys Trp Leu Phe Leu Phe Ile 215 220 225 230 gga tgg tgc ataccc tgc cct atc att gtc gcc tgg gca gtt ggc aaa 953 Gly Trp Cys Ile ProCys Pro Ile Ile Val Ala Trp Ala Val Gly Lys 235 240 245 ctc tac tat gagaat gag cag tgc tgg ttt ggc aag gaa cct ggt gac 1001 Leu Tyr Tyr Glu AsnGlu Gln Cys Trp Phe Gly Lys Glu Pro Gly Asp 250 255 260 tta gtg gac tacatc tac cag ggc ccc atc atc ctc gtg ctc ctc atc 1049 Leu Val Asp Tyr IleTyr Gln Gly Pro Ile Ile Leu Val Leu Leu Ile 265 270 275 aat ttt gtg tttctg ttc aac atc gtc agg atc ctg atg aca aaa ctg 1097 Asn Phe Val Phe LeuPhe Asn Ile Val Arg Ile Leu Met Thr Lys Leu 280 285 290 cga gcc tcc accaca tcc gag acc atc cag tac agg aag gca gtg aag 1145 Arg Ala Ser Thr ThrSer Glu Thr Ile Gln Tyr Arg Lys Ala Val Lys 295 300 305 310 gcc acc ctggtc ctc ctc ccc ctg ttg ggc atc acc tac atg ctc ttc 1193 Ala Thr Leu ValLeu Leu Pro Leu Leu Gly Ile Thr Tyr Met Leu Phe 315 320 325 ttt gtc aatcct gga gag gac gac ctg tca cag att gtg ttc atc tac 1241 Phe Val Asn ProGly Glu Asp Asp Leu Ser Gln Ile Val Phe Ile Tyr 330 335 340 ttc aac tctttc ctg cag tcc ttt cag ggt ttc ttt gtg tcc gtt ttc 1289 Phe Asn Ser PheLeu Gln Ser Phe Gln Gly Phe Phe Val Ser Val Phe 345 350 355 tac tgc ttcttc aat gga gag gtg cgc tcc gcc ctg aga aag cgg tgg 1337 Tyr Cys Phe PheAsn Gly Glu Val Arg Ser Ala Leu Arg Lys Arg Trp 360 365 370 cac cgt tggcag gac cac cac gcc ctc cga gtg cct gtg gcc cgg gcc 1385 His Arg Trp GlnAsp His His Ala Leu Arg Val Pro Val Ala Arg Ala 375 380 385 390 atg tccatt ccc aca tcg ccc acc agg atc agc ttc cac agc atc aag 1433 Met Ser IlePro Thr Ser Pro Thr Arg Ile Ser Phe His Ser Ile Lys 395 400 405 cag acagct gcc gtg tga tcccctgtca cccatctgcc cagcactcca 1481 Gln Thr Ala AlaVal 410 ccaccgaggc ggcttcctca ttcttcacag ccttccctgg gtcctccttgctacactgac 1541 ccttgggtgc aggagaaggg ggggtggatg aactctcctg ccggaagaaaggaaaactat 1601 gaaatggagg ctctgaaaga ccagg 1626 18 411 PRT Rattusnorvegicus 18 Met Asp Ala Ala Leu Leu Leu Ser Leu Leu Glu Ala Asn CysSer Leu 1 5 10 15 Ala Leu Ala Glu Glu Leu Leu Leu Asp Gly Trp Gly GluPro Pro Asp 20 25 30 Pro Glu Gly Pro Tyr Ser Tyr Cys Asn Thr Thr Leu AspGln Ile Gly 35 40 45 Thr Cys Trp Pro Gln Ser Ala Pro Gly Ala Leu Val GluArg Pro Cys 50 55 60 Pro Glu Tyr Phe Asn Gly Ile Lys Tyr Asn Thr Thr ArgAsn Ala Tyr 65 70 75 80 Arg Glu Cys Leu Glu Asn Gly Thr Trp Ala Ser ArgIle Asn Tyr Ser 85 90 95 His Cys Glu Pro Ile Leu Asp Asp Lys Gln Arg LysTyr Asp Leu His 100 105 110 Tyr Arg Ile Ala Leu Ile Ile Asn Tyr Leu GlyHis Cys Val Ser Val 115 120 125 Val Ala Leu Val Ala Ala Phe Leu Leu PheLeu Val Leu Arg Ser Ile 130 135 140 Arg Cys Leu Arg Asn Val Ile His TrpAsn Leu Ile Thr Thr Phe Ile 145 150 155 160 Leu Arg Asn Ile Thr Trp PheLeu Leu Gln Leu Ile Asp His Glu Val 165 170 175 His Glu Gly Asn Glu ValTrp Cys Arg Cys Val Thr Thr Ile Phe Asn 180 185 190 Tyr Phe Val Val ThrAsn Phe Phe Trp Met Phe Val Glu Gly Cys Tyr 195 200 205 Leu His Thr AlaIle Val Met Thr Tyr Ser Thr Glu His Leu Arg Lys 210 215 220 Trp Leu PheLeu Phe Ile Gly Trp Cys Ile Pro Cys Pro Ile Ile Val 225 230 235 240 AlaTrp Ala Val Gly Lys Leu Tyr Tyr Glu Asn Glu Gln Cys Trp Phe 245 250 255Gly Lys Glu Pro Gly Asp Leu Val Asp Tyr Ile Tyr Gln Gly Pro Ile 260 265270 Ile Leu Val Leu Leu Ile Asn Phe Val Phe Leu Phe Asn Ile Val Arg 275280 285 Ile Leu Met Thr Lys Leu Arg Ala Ser Thr Thr Ser Glu Thr Ile Gln290 295 300 Tyr Arg Lys Ala Val Lys Ala Thr Leu Val Leu Leu Pro Leu LeuGly 305 310 315 320 Ile Thr Tyr Met Leu Phe Phe Val Asn Pro Gly Glu AspAsp Leu Ser 325 330 335 Gln Ile Val Phe Ile Tyr Phe Asn Ser Phe Leu GlnSer Phe Gln Gly 340 345 350 Phe Phe Val Ser Val Phe Tyr Cys Phe Phe AsnGly Glu Val Arg Ser 355 360 365 Ala Leu Arg Lys Arg Trp His Arg Trp GlnAsp His His Ala Leu Arg 370 375 380 Val Pro Val Ala Arg Ala Met Ser IlePro Thr Ser Pro Thr Arg Ile 385 390 395 400 Ser Phe His Ser Ile Lys GlnThr Ala Ala Val 405 410 19 1514 DNA Rattus norvegicus CDS (44)..(1339)19 gatccctatc cctgagcaag cgagtggcag gatctggtgt ccc atg ggg cac cca 55Met Gly His Pro 1 ggc tct ctt ccc agt gca caa ctc ctc ctc tgc cta tactct ctg ctc 103 Gly Ser Leu Pro Ser Ala Gln Leu Leu Leu Cys Leu Tyr SerLeu Leu 5 10 15 20 cca ctg ctc cag gtg gcc caa cca ggc agg cca ctc caggac cag ccc 151 Pro Leu Leu Gln Val Ala Gln Pro Gly Arg Pro Leu Gln AspGln Pro 25 30 35 ctg tgg aca ctt ttg gag cag tac tgc cat agg acc aca actcgg aat 199 Leu Trp Thr Leu Leu Glu Gln Tyr Cys His Arg Thr Thr Thr ArgAsn 40 45 50 ttt tca ggt ccc tac tcc tac tgc tac acg acc ttg gac cag atcggg 247 Phe Ser Gly Pro Tyr Ser Tyr Cys Tyr Thr Thr Leu Asp Gln Ile Gly55 60 65 acc tgc tgg ccc cag agc gcg cct gga gcc cta gtg gag aga cca tgc295 Thr Cys Trp Pro Gln Ser Ala Pro Gly Ala Leu Val Glu Arg Pro Cys 7075 80 ccc gaa tac ttc aac ggc atc aag tac aac acg acc cgg aat gcc tac343 Pro Glu Tyr Phe Asn Gly Ile Lys Tyr Asn Thr Thr Arg Asn Ala Tyr 8590 95 100 aga gaa tgc ctg gag aat ggg acc tgg gcc tca agg atc aac tactca 391 Arg Glu Cys Leu Glu Asn Gly Thr Trp Ala Ser Arg Ile Asn Tyr Ser105 110 115 cac tgt gaa ccc att ttg gat gac aag cag agg aag tat gac ctgcat 439 His Cys Glu Pro Ile Leu Asp Asp Lys Gln Arg Lys Tyr Asp Leu His120 125 130 tac cga atc gcc ctc atc atc aac tac ctg ggc cac tgt gtt tccgtg 487 Tyr Arg Ile Ala Leu Ile Ile Asn Tyr Leu Gly His Cys Val Ser Val135 140 145 gtg gcc ctg gtg gct gct ttc ctg ctt ttc cta gtg ctg cgg agtatc 535 Val Ala Leu Val Ala Ala Phe Leu Leu Phe Leu Val Leu Arg Ser Ile150 155 160 cgc tgc ctg cgg aat gtg atc cac tgg aac ctc atc acc acc ttcatc 583 Arg Cys Leu Arg Asn Val Ile His Trp Asn Leu Ile Thr Thr Phe Ile165 170 175 180 ctg aga aac atc acg tgg ttc ctg ctg caa ctc atc gac cacgaa gtg 631 Leu Arg Asn Ile Thr Trp Phe Leu Leu Gln Leu Ile Asp His GluVal 185 190 195 cat gag ggc aat gag gtc tgg tgc cgc tgc gtc acc acc atattc aac 679 His Glu Gly Asn Glu Val Trp Cys Arg Cys Val Thr Thr Ile PheAsn 200 205 210 tac ttt gtg gtc acc aac ttc ttc tgg atg ttt gtg gaa ggctgc tac 727 Tyr Phe Val Val Thr Asn Phe Phe Trp Met Phe Val Glu Gly CysTyr 215 220 225 ctg cac acg gcc atc gtc atg acg tac tcc acg gag cat ctgcgc aag 775 Leu His Thr Ala Ile Val Met Thr Tyr Ser Thr Glu His Leu ArgLys 230 235 240 tgg ctc ttc ctc ttc att gga tgg tgc ata ccc tgc cct atcatt gtc 823 Trp Leu Phe Leu Phe Ile Gly Trp Cys Ile Pro Cys Pro Ile IleVal 245 250 255 260 gcc tgg gca gtt ggc aaa ctc tac tat gag aat gag cagtgc tgg ttt 871 Ala Trp Ala Val Gly Lys Leu Tyr Tyr Glu Asn Glu Gln CysTrp Phe 265 270 275 ggc aag gaa cct ggt gac tta gtg gac tac atc tac cagggc ccc atc 919 Gly Lys Glu Pro Gly Asp Leu Val Asp Tyr Ile Tyr Gln GlyPro Ile 280 285 290 atc ctc gtg ctc ctc atc aat ttt gtg ttt ctg ttc aacatc gtc agg 967 Ile Leu Val Leu Leu Ile Asn Phe Val Phe Leu Phe Asn IleVal Arg 295 300 305 atc ctg atg aca aaa ctg cga gcc tcc acc aca tcc gagacc atc cag 1015 Ile Leu Met Thr Lys Leu Arg Ala Ser Thr Thr Ser Glu ThrIle Gln 310 315 320 tac agg aag gca gtg aag gcc aac ctg gtc ctc ctc cccctg ttg ggc 1063 Tyr Arg Lys Ala Val Lys Ala Asn Leu Val Leu Leu Pro LeuLeu Gly 325 330 335 340 atc acc tac atg ctc ttc ttt gtc aat cct gga gaggac gac ctg tca 1111 Ile Thr Tyr Met Leu Phe Phe Val Asn Pro Gly Glu AspAsp Leu Ser 345 350 355 cag att gtg ttc atc tac ttc aac tct ttc ctg cagtcc ttt cag ggt 1159 Gln Ile Val Phe Ile Tyr Phe Asn Ser Phe Leu Gln SerPhe Gln Gly 360 365 370 ttc ttt gtg tcc gtt ttc tac tgc ttc ttc aat ggagag gtg cgc tcc 1207 Phe Phe Val Ser Val Phe Tyr Cys Phe Phe Asn Gly GluVal Arg Ser 375 380 385 gcc ctg aga aag cgg tgg cac cgt tgg cag gac caccac gcc ctc cga 1255 Ala Leu Arg Lys Arg Trp His Arg Trp Gln Asp His HisAla Leu Arg 390 395 400 gtg cct gtg gcc cgg gcc atg tcc att ccc aca tcgccc acc agg atc 1303 Val Pro Val Ala Arg Ala Met Ser Ile Pro Thr Ser ProThr Arg Ile 405 410 415 420 agc ttc cac agc atc aag cag aca gct gcc gtgtga tcccctgtca 1349 Ser Phe His Ser Ile Lys Gln Thr Ala Ala Val 425 430cccatctgcc cagcactcca ccaccgaggc ggcttcctca ttcttcacag ccttccctgg 1409gtcctccttg ctacactgac ccttgggtgc aggagaaggg ggggtggatg aactctcctg 1469ccggaagaaa ggaaaactat gaaatggagg ctctgaaaga ccagg 1514 20 431 PRT Rattusnorvegicus 20 Met Gly His Pro Gly Ser Leu Pro Ser Ala Gln Leu Leu LeuCys Leu 1 5 10 15 Tyr Ser Leu Leu Pro Leu Leu Gln Val Ala Gln Pro GlyArg Pro Leu 20 25 30 Gln Asp Gln Pro Leu Trp Thr Leu Leu Glu Gln Tyr CysHis Arg Thr 35 40 45 Thr Thr Arg Asn Phe Ser Gly Pro Tyr Ser Tyr Cys TyrThr Thr Leu 50 55 60 Asp Gln Ile Gly Thr Cys Trp Pro Gln Ser Ala Pro GlyAla Leu Val 65 70 75 80 Glu Arg Pro Cys Pro Glu Tyr Phe Asn Gly Ile LysTyr Asn Thr Thr 85 90 95 Arg Asn Ala Tyr Arg Glu Cys Leu Glu Asn Gly ThrTrp Ala Ser Arg 100 105 110 Ile Asn Tyr Ser His Cys Glu Pro Ile Leu AspAsp Lys Gln Arg Lys 115 120 125 Tyr Asp Leu His Tyr Arg Ile Ala Leu IleIle Asn Tyr Leu Gly His 130 135 140 Cys Val Ser Val Val Ala Leu Val AlaAla Phe Leu Leu Phe Leu Val 145 150 155 160 Leu Arg Ser Ile Arg Cys LeuArg Asn Val Ile His Trp Asn Leu Ile 165 170 175 Thr Thr Phe Ile Leu ArgAsn Ile Thr Trp Phe Leu Leu Gln Leu Ile 180 185 190 Asp His Glu Val HisGlu Gly Asn Glu Val Trp Cys Arg Cys Val Thr 195 200 205 Thr Ile Phe AsnTyr Phe Val Val Thr Asn Phe Phe Trp Met Phe Val 210 215 220 Glu Gly CysTyr Leu His Thr Ala Ile Val Met Thr Tyr Ser Thr Glu 225 230 235 240 HisLeu Arg Lys Trp Leu Phe Leu Phe Ile Gly Trp Cys Ile Pro Cys 245 250 255Pro Ile Ile Val Ala Trp Ala Val Gly Lys Leu Tyr Tyr Glu Asn Glu 260 265270 Gln Cys Trp Phe Gly Lys Glu Pro Gly Asp Leu Val Asp Tyr Ile Tyr 275280 285 Gln Gly Pro Ile Ile Leu Val Leu Leu Ile Asn Phe Val Phe Leu Phe290 295 300 Asn Ile Val Arg Ile Leu Met Thr Lys Leu Arg Ala Ser Thr ThrSer 305 310 315 320 Glu Thr Ile Gln Tyr Arg Lys Ala Val Lys Ala Asn LeuVal Leu Leu 325 330 335 Pro Leu Leu Gly Ile Thr Tyr Met Leu Phe Phe ValAsn Pro Gly Glu 340 345 350 Asp Asp Leu Ser Gln Ile Val Phe Ile Tyr PheAsn Ser Phe Leu Gln 355 360 365 Ser Phe Gln Gly Phe Phe Val Ser Val PheTyr Cys Phe Phe Asn Gly 370 375 380 Glu Val Arg Ser Ala Leu Arg Lys ArgTrp His Arg Trp Gln Asp His 385 390 395 400 His Ala Leu Arg Val Pro ValAla Arg Ala Met Ser Ile Pro Thr Ser 405 410 415 Pro Thr Arg Ile Ser PheHis Ser Ile Lys Gln Thr Ala Ala Val 420 425 430 21 2273 DNA Mus musculusCDS (32)..(1279) 21 tatccaggac gctgacagag cgagcccgag g atg gga cag cgcccg cag ctc 52 Met Gly Gln Arg Pro Gln Leu 1 5 cgg ctc gtg aag gcc cttctc ctt ctg ggg ctg aac ccc gtc tcc acc 100 Arg Leu Val Lys Ala Leu LeuLeu Leu Gly Leu Asn Pro Val Ser Thr 10 15 20 tcc ctc cag gat cag cag tgtgag agc ctg tcc ctg gcc agc aat gtc 148 Ser Leu Gln Asp Gln Gln Cys GluSer Leu Ser Leu Ala Ser Asn Val 25 30 35 tct ggc ctg cag tgc aat gcc tccgtg gac ctc att ggc acc tgc tgg 196 Ser Gly Leu Gln Cys Asn Ala Ser ValAsp Leu Ile Gly Thr Cys Trp 40 45 50 55 ccc agg agc cct gca ggg cag ttggtg gtt cgg ccc tgc cct gcc ttt 244 Pro Arg Ser Pro Ala Gly Gln Leu ValVal Arg Pro Cys Pro Ala Phe 60 65 70 ttc tac ggt gtc cgc tac aac acc acaaac aat ggc tac cgg gaa tgc 292 Phe Tyr Gly Val Arg Tyr Asn Thr Thr AsnAsn Gly Tyr Arg Glu Cys 75 80 85 ctg gcc aac ggc agc tgg gca gcc cgt gtgaat tat tct gag tgc cag 340 Leu Ala Asn Gly Ser Trp Ala Ala Arg Val AsnTyr Ser Glu Cys Gln 90 95 100 gag att ctc aac gaa gag aag aag agc aaagtg cac tac cac att gcc 388 Glu Ile Leu Asn Glu Glu Lys Lys Ser Lys ValHis Tyr His Ile Ala 105 110 115 gtc atc atc aac tac ctg ggc cac tgc atctcc ctg gtg gcc ctc ctg 436 Val Ile Ile Asn Tyr Leu Gly His Cys Ile SerLeu Val Ala Leu Leu 120 125 130 135 gtg gcc ttt gtc ctc ttc ctg cgg ctcagg agc atc cgg tgc ctg agg 484 Val Ala Phe Val Leu Phe Leu Arg Leu ArgSer Ile Arg Cys Leu Arg 140 145 150 aac atc atc cac tgg aac ctc atc tcggct ttc atc ctg cgc aac gcc 532 Asn Ile Ile His Trp Asn Leu Ile Ser AlaPhe Ile Leu Arg Asn Ala 155 160 165 acg tgg ttt gtg gtc cag ctc acc gtgagc ccc gag gtc cac cag agc 580 Thr Trp Phe Val Val Gln Leu Thr Val SerPro Glu Val His Gln Ser 170 175 180 aac gtg gcc tgg tgc agg ctg gtg acagcc gcc tac aac tac ttc cac 628 Asn Val Ala Trp Cys Arg Leu Val Thr AlaAla Tyr Asn Tyr Phe His 185 190 195 gta acc aac ttc ttc tgg atg ttc ggtgag ggc tgc tac ctg cac aca 676 Val Thr Asn Phe Phe Trp Met Phe Gly GluGly Cys Tyr Leu His Thr 200 205 210 215 gcc atc gta ctc acg tac tcc accgac cgt ctg cgc aag tgg atg ttc 724 Ala Ile Val Leu Thr Tyr Ser Thr AspArg Leu Arg Lys Trp Met Phe 220 225 230 gtc tgc atc ggc tgg ggt gtg cctttc ccc atc att gtg gct tgg gcc 772 Val Cys Ile Gly Trp Gly Val Pro PhePro Ile Ile Val Ala Trp Ala 235 240 245 att ggg aaa ctt tac tac gac aatgaa aag tgc tgg ttt ggc aaa cgt 820 Ile Gly Lys Leu Tyr Tyr Asp Asn GluLys Cys Trp Phe Gly Lys Arg 250 255 260 cct gga gta tat act gac tac atctac cag ggc ccc atg atc ctg gtc 868 Pro Gly Val Tyr Thr Asp Tyr Ile TyrGln Gly Pro Met Ile Leu Val 265 270 275 ctg ctg atc aac ttt atc ttt ctcttc aac att gtc cgc atc ctc atg 916 Leu Leu Ile Asn Phe Ile Phe Leu PheAsn Ile Val Arg Ile Leu Met 280 285 290 295 acc aaa ctc cga gca tcc accaca tct gag act att cag tac agg aag 964 Thr Lys Leu Arg Ala Ser Thr ThrSer Glu Thr Ile Gln Tyr Arg Lys 300 305 310 gct gtg aag gcc act ctg gtgctc ttg ccc ctc ctg ggc atc acc tac 1012 Ala Val Lys Ala Thr Leu Val LeuLeu Pro Leu Leu Gly Ile Thr Tyr 315 320 325 atg ttg ttc ttc gtc aac cctggg gag gac gag gtc tcc agg gtt gtc 1060 Met Leu Phe Phe Val Asn Pro GlyGlu Asp Glu Val Ser Arg Val Val 330 335 340 ttc atc tac ttc aac tct ttcctg gag tcc ttt cag ggc ttc ttc gtg 1108 Phe Ile Tyr Phe Asn Ser Phe LeuGlu Ser Phe Gln Gly Phe Phe Val 345 350 355 tct gtg ttc tat tgt ttt ctgaac agt gag gtc cgc tct gcc atc cgg 1156 Ser Val Phe Tyr Cys Phe Leu AsnSer Glu Val Arg Ser Ala Ile Arg 360 365 370 375 aag agg tgg cgg cga tggcag gac aag cac tcc atc aga gcc cga gtg 1204 Lys Arg Trp Arg Arg Trp GlnAsp Lys His Ser Ile Arg Ala Arg Val 380 385 390 gcc cgc gcc atg tcc atcccc acc tcc ccc acc aga gtc agc ttc cac 1252 Ala Arg Ala Met Ser Ile ProThr Ser Pro Thr Arg Val Ser Phe His 395 400 405 agc atc aag cag tcc acagca gtg tga acctcaggcc acagagcagc 1299 Ser Ile Lys Gln Ser Thr Ala Val410 415 ccccaagacc cgaggctggg gaaatgatgc aagctcacta gcgagcctgtctgcagaggc 1359 aggcagcctt cccatccctg cccctgggat gcagacctgt aagcctgcccagccgtggac 1419 aaagcccata gcactggggt gggcccttgg catctagctc cctgctgcccattctccctg 1479 ggaagttgaa atgggcattg ggggctggaa accctgcagc agtttgatgggcctgtgagc 1539 gctgtcttct cccagagcag cttactgaag atctgtcgtc tccaggagctgttggggagg 1599 ccaactgtta ccctggggca tcatggaaaa ctcccttctg agactgtaaagtctctgagt 1659 gttagcgatg ccttgggatg ctaccgagga ccaacatggt ccagtcaggagaccgggaga 1719 tagcggtaga aatctgggaa cgtcatcaga tggcactcca cctccctacaagtcactcct 1779 gagccaccag gatttcatca gcactgtggc actgccactg gaaagccctgccttgctgct 1839 ttgctgccct gcacctttag acatttacta ttctgcaggc caagccagctttctgtcact 1899 22 415 PRT Mus musculus 22 Met Gly Gln Arg Pro Gln LeuArg Leu Val Lys Ala Leu Leu Leu Leu 1 5 10 15 Gly Leu Asn Pro Val SerThr Ser Leu Gln Asp Gln Gln Cys Glu Ser 20 25 30 Leu Ser Leu Ala Ser AsnVal Ser Gly Leu Gln Cys Asn Ala Ser Val 35 40 45 Asp Leu Ile Gly Thr CysTrp Pro Arg Ser Pro Ala Gly Gln Leu Val 50 55 60 Val Arg Pro Cys Pro AlaPhe Phe Tyr Gly Val Arg Tyr Asn Thr Thr 65 70 75 80 Asn Asn Gly Tyr ArgGlu Cys Leu Ala Asn Gly Ser Trp Ala Ala Arg 85 90 95 Val Asn Tyr Ser GluCys Gln Glu Ile Leu Asn Glu Glu Lys Lys Ser 100 105 110 Lys Val His TyrHis Ile Ala Val Ile Ile Asn Tyr Leu Gly His Cys 115 120 125 Ile Ser LeuVal Ala Leu Leu Val Ala Phe Val Leu Phe Leu Arg Leu 130 135 140 Arg SerIle Arg Cys Leu Arg Asn Ile Ile His Trp Asn Leu Ile Ser 145 150 155 160Ala Phe Ile Leu Arg Asn Ala Thr Trp Phe Val Val Gln Leu Thr Val 165 170175 Ser Pro Glu Val His Gln Ser Asn Val Ala Trp Cys Arg Leu Val Thr 180185 190 Ala Ala Tyr Asn Tyr Phe His Val Thr Asn Phe Phe Trp Met Phe Gly195 200 205 Glu Gly Cys Tyr Leu His Thr Ala Ile Val Leu Thr Tyr Ser ThrAsp 210 215 220 Arg Leu Arg Lys Trp Met Phe Val Cys Ile Gly Trp Gly ValPro Phe 225 230 235 240 Pro Ile Ile Val Ala Trp Ala Ile Gly Lys Leu TyrTyr Asp Asn Glu 245 250 255 Lys Cys Trp Phe Gly Lys Arg Pro Gly Val TyrThr Asp Tyr Ile Tyr 260 265 270 Gln Gly Pro Met Ile Leu Val Leu Leu IleAsn Phe Ile Phe Leu Phe 275 280 285 Asn Ile Val Arg Ile Leu Met Thr LysLeu Arg Ala Ser Thr Thr Ser 290 295 300 Glu Thr Ile Gln Tyr Arg Lys AlaVal Lys Ala Thr Leu Val Leu Leu 305 310 315 320 Pro Leu Leu Gly Ile ThrTyr Met Leu Phe Phe Val Asn Pro Gly Glu 325 330 335 Asp Glu Val Ser ArgVal Val Phe Ile Tyr Phe Asn Ser Phe Leu Glu 340 345 350 Ser Phe Gln GlyPhe Phe Val Ser Val Phe Tyr Cys Phe Leu Asn Ser 355 360 365 Glu Val ArgSer Ala Ile Arg Lys Arg Trp Arg Arg Trp Gln Asp Lys 370 375 380 His SerIle Arg Ala Arg Val Ala Arg Ala Met Ser Ile Pro Thr Ser 385 390 395 400Pro Thr Arg Val Ser Phe His Ser Ile Lys Gln Ser Thr Ala Val 405 410 41523 1374 DNA Mus musculus CDS (79)..(1374) 23 gccggacaga cctcctttggaagcagccac ttctggtccc catccctgga gcgatcgagc 60 ggcaggatct gctgtccc atgggg acc cca ggc tct ctt ccc agt gca cag 111 Met Gly Thr Pro Gly Ser LeuPro Ser Ala Gln 1 5 10 ctt ctc ctc tgc ctg ttt tcc ctg ctt cca gtg ctccag gtg gcc caa 159 Leu Leu Leu Cys Leu Phe Ser Leu Leu Pro Val Leu GlnVal Ala Gln 15 20 25 cca ggc cag gca ccc cag gac cag ccc ctg tgg aca cttttg gag cag 207 Pro Gly Gln Ala Pro Gln Asp Gln Pro Leu Trp Thr Leu LeuGlu Gln 30 35 40 tac tgc cac agg acc aca att ggg aat ttt tca ggt ccc tacacc tac 255 Tyr Cys His Arg Thr Thr Ile Gly Asn Phe Ser Gly Pro Tyr ThrTyr 45 50 55 tgc aac acg acc ttg gac cag atc ggg acc tgc tgg cca cag agcgca 303 Cys Asn Thr Thr Leu Asp Gln Ile Gly Thr Cys Trp Pro Gln Ser Ala60 65 70 75 ccc gga gcc cta gta gag aga ccg tgc ccc gag tac ttc aat ggcatc 351 Pro Gly Ala Leu Val Glu Arg Pro Cys Pro Glu Tyr Phe Asn Gly Ile80 85 90 aag tac aac acg acc cgg aat gcc tac aga gag tgc ctg gag aac ggg399 Lys Tyr Asn Thr Thr Arg Asn Ala Tyr Arg Glu Cys Leu Glu Asn Gly 95100 105 acc tgg gcc tca agg gtc aac tac tca cac tgc gaa ccc att ttg gat447 Thr Trp Ala Ser Arg Val Asn Tyr Ser His Cys Glu Pro Ile Leu Asp 110115 120 gac aag cag aga aag tat gac ctg cat tac cga atc gcc ctc att gtc495 Asp Lys Gln Arg Lys Tyr Asp Leu His Tyr Arg Ile Ala Leu Ile Val 125130 135 aac tac ctg ggt cac tgt gtt tcc gtg gtg gcc ctg gtg gcc gct ttc543 Asn Tyr Leu Gly His Cys Val Ser Val Val Ala Leu Val Ala Ala Phe 140145 150 155 ctg ctt ttc cta gtg ctg cgg agt atc cgc tgc ctg agg aat gtgatc 591 Leu Leu Phe Leu Val Leu Arg Ser Ile Arg Cys Leu Arg Asn Val Ile160 165 170 cac tgg aac ctc atc acc acc ttc att ctg aga aac atc gcg tggttc 639 His Trp Asn Leu Ile Thr Thr Phe Ile Leu Arg Asn Ile Ala Trp Phe175 180 185 ctg ctg caa ctc atc gac cac gaa gtg cac gag ggc aat gag gtctgg 687 Leu Leu Gln Leu Ile Asp His Glu Val His Glu Gly Asn Glu Val Trp190 195 200 tgc cgc tgc atc acc acc atc ttc aac tat ttt gtg gtc acc aacttc 735 Cys Arg Cys Ile Thr Thr Ile Phe Asn Tyr Phe Val Val Thr Asn Phe205 210 215 ttc tgg atg ttt gtg gag ggc tgc tac ctg cac acg gcc att gtcatg 783 Phe Trp Met Phe Val Glu Gly Cys Tyr Leu His Thr Ala Ile Val Met220 225 230 235 acg tac tcc aca gag cac ctg cgc aag tgg ctt ttc ctc ttcatt gga 831 Thr Tyr Ser Thr Glu His Leu Arg Lys Trp Leu Phe Leu Phe IleGly 240 245 250 tgg tgc att ccc tgc cct atc atc atc gcc tgg gca gtt ggcaaa ctc 879 Trp Cys Ile Pro Cys Pro Ile Ile Ile Ala Trp Ala Val Gly LysLeu 255 260 265 tac tat gag aat gag cag tgc tgg ttt ggc aag gaa gct ggtgat ttg 927 Tyr Tyr Glu Asn Glu Gln Cys Trp Phe Gly Lys Glu Ala Gly AspLeu 270 275 280 gtg gac tac atc tac cag ggc ccc gtc atg ctt gtg ctg ttgatc aat 975 Val Asp Tyr Ile Tyr Gln Gly Pro Val Met Leu Val Leu Leu IleAsn 285 290 295 ttt gta ttt ctg ttt aac atc gtc agg atc ctg atg acg aagtta cga 1023 Phe Val Phe Leu Phe Asn Ile Val Arg Ile Leu Met Thr Lys LeuArg 300 305 310 315 gca tcc acc acg tcc gag aca atc caa tac agg aag gcagtg aag gcc 1071 Ala Ser Thr Thr Ser Glu Thr Ile Gln Tyr Arg Lys Ala ValLys Ala 320 325 330 acg ctg gtc ctc ctc ccc ctg ttg ggc atc acc tac atgctc ttc ttt 1119 Thr Leu Val Leu Leu Pro Leu Leu Gly Ile Thr Tyr Met LeuPhe Phe 335 340 345 gtc aat cct ggc gag gac gac ctg tcc cag att gtg ttcatc tac ttc 1167 Val Asn Pro Gly Glu Asp Asp Leu Ser Gln Ile Val Phe IleTyr Phe 350 355 360 aac tct ttc ctg cag tcc ttc cag ggt ttc ttt gtg tccgtt ttc tac 1215 Asn Ser Phe Leu Gln Ser Phe Gln Gly Phe Phe Val Ser ValPhe Tyr 365 370 375 tgc ttc ttc aat gga gag gtg cgc gcg gcc ctg aga aagcgg tgg cac 1263 Cys Phe Phe Asn Gly Glu Val Arg Ala Ala Leu Arg Lys ArgTrp His 380 385 390 395 cgc tgg cag gac cac cac gcc ctc cgg gtg cct gtggcc cgg gcc atg 1311 Arg Trp Gln Asp His His Ala Leu Arg Val Pro Val AlaArg Ala Met 400 405 410 tcc atc cct acg tcg ccc acc agg atc agc ttc cacagc atc aag cag 1359 Ser Ile Pro Thr Ser Pro Thr Arg Ile Ser Phe His SerIle Lys Gln 415 420 425 aca gct gct gtg tga 1374 Thr Ala Ala Val 430 24431 PRT Mus musculus 24 Met Gly Thr Pro Gly Ser Leu Pro Ser Ala Gln LeuLeu Leu Cys Leu 1 5 10 15 Phe Ser Leu Leu Pro Val Leu Gln Val Ala GlnPro Gly Gln Ala Pro 20 25 30 Gln Asp Gln Pro Leu Trp Thr Leu Leu Glu GlnTyr Cys His Arg Thr 35 40 45 Thr Ile Gly Asn Phe Ser Gly Pro Tyr Thr TyrCys Asn Thr Thr Leu 50 55 60 Asp Gln Ile Gly Thr Cys Trp Pro Gln Ser AlaPro Gly Ala Leu Val 65 70 75 80 Glu Arg Pro Cys Pro Glu Tyr Phe Asn GlyIle Lys Tyr Asn Thr Thr 85 90 95 Arg Asn Ala Tyr Arg Glu Cys Leu Glu AsnGly Thr Trp Ala Ser Arg 100 105 110 Val Asn Tyr Ser His Cys Glu Pro IleLeu Asp Asp Lys Gln Arg Lys 115 120 125 Tyr Asp Leu His Tyr Arg Ile AlaLeu Ile Val Asn Tyr Leu Gly His 130 135 140 Cys Val Ser Val Val Ala LeuVal Ala Ala Phe Leu Leu Phe Leu Val 145 150 155 160 Leu Arg Ser Ile ArgCys Leu Arg Asn Val Ile His Trp Asn Leu Ile 165 170 175 Thr Thr Phe IleLeu Arg Asn Ile Ala Trp Phe Leu Leu Gln Leu Ile 180 185 190 Asp His GluVal His Glu Gly Asn Glu Val Trp Cys Arg Cys Ile Thr 195 200 205 Thr IlePhe Asn Tyr Phe Val Val Thr Asn Phe Phe Trp Met Phe Val 210 215 220 GluGly Cys Tyr Leu His Thr Ala Ile Val Met Thr Tyr Ser Thr Glu 225 230 235240 His Leu Arg Lys Trp Leu Phe Leu Phe Ile Gly Trp Cys Ile Pro Cys 245250 255 Pro Ile Ile Ile Ala Trp Ala Val Gly Lys Leu Tyr Tyr Glu Asn Glu260 265 270 Gln Cys Trp Phe Gly Lys Glu Ala Gly Asp Leu Val Asp Tyr IleTyr 275 280 285 Gln Gly Pro Val Met Leu Val Leu Leu Ile Asn Phe Val PheLeu Phe 290 295 300 Asn Ile Val Arg Ile Leu Met Thr Lys Leu Arg Ala SerThr Thr Ser 305 310 315 320 Glu Thr Ile Gln Tyr Arg Lys Ala Val Lys AlaThr Leu Val Leu Leu 325 330 335 Pro Leu Leu Gly Ile Thr Tyr Met Leu PhePhe Val Asn Pro Gly Glu 340 345 350 Asp Asp Leu Ser Gln Ile Val Phe IleTyr Phe Asn Ser Phe Leu Gln 355 360 365 Ser Phe Gln Gly Phe Phe Val SerVal Phe Tyr Cys Phe Phe Asn Gly 370 375 380 Glu Val Arg Ala Ala Leu ArgLys Arg Trp His Arg Trp Gln Asp His 385 390 395 400 His Ala Leu Arg ValPro Val Ala Arg Ala Met Ser Ile Pro Thr Ser 405 410 415 Pro Thr Arg IleSer Phe His Ser Ile Lys Gln Thr Ala Ala Val 420 425 430 25 2617 DNA Musmusculus CDS (132)..(1424) 25 gggcattacc ttggtgggta ggtcgggcagggtaggacag gcctaagaga gaggccggac 60 agacctcctt tggaagcagc cacttctggtccccatccct ggagcgatcg agcggcagga 120 tctgctgtcc c atg ggg acc cca ggctct ctt ccc agt gca cag ctt ctc 170 Met Gly Thr Pro Gly Ser Leu Pro SerAla Gln Leu Leu 1 5 10 ctc tgc ctg ttt tcc ctg ctt cca gtg ctc cag gtggcc caa cca ggc 218 Leu Cys Leu Phe Ser Leu Leu Pro Val Leu Gln Val AlaGln Pro Gly 15 20 25 cag gca ccc cag gac cag ccc ctg tgg aca ctt ttg gagcag tac tgc 266 Gln Ala Pro Gln Asp Gln Pro Leu Trp Thr Leu Leu Glu GlnTyr Cys 30 35 40 45 cac agg acc aca att ggg aat ttt tca ggt ccc tac acctac tgc aac 314 His Arg Thr Thr Ile Gly Asn Phe Ser Gly Pro Tyr Thr TyrCys Asn 50 55 60 acg acc ttg gac cag atc ggg acc tgc tgg cca cag agc gcaccc gga 362 Thr Thr Leu Asp Gln Ile Gly Thr Cys Trp Pro Gln Ser Ala ProGly 65 70 75 gcc cta gta gag aga ccg tgc ccc gag tac ttc aat ggc atc aagtac 410 Ala Leu Val Glu Arg Pro Cys Pro Glu Tyr Phe Asn Gly Ile Lys Tyr80 85 90 aac acg acc cgg aat gcc tac aga gag tgc ctg gag aac ggg acc tgg458 Asn Thr Thr Arg Asn Ala Tyr Arg Glu Cys Leu Glu Asn Gly Thr Trp 95100 105 gcc tca agg gtc aac tac tca cac tgc gaa ccc att ttg gat gac aag506 Ala Ser Arg Val Asn Tyr Ser His Cys Glu Pro Ile Leu Asp Asp Lys 110115 120 125 aga aag tat gac ctg cat tac cga atc gcc ctc att gtc aac tacctg 554 Arg Lys Tyr Asp Leu His Tyr Arg Ile Ala Leu Ile Val Asn Tyr Leu130 135 140 ggt cac tgt gtt tcc gtg gtg gcc ctg gtg gcc gct ttc ctg cttttc 602 Gly His Cys Val Ser Val Val Ala Leu Val Ala Ala Phe Leu Leu Phe145 150 155 cta gtg ctg cgg agt atc cgc tgc ctg agg aat gtg atc cac tggaac 650 Leu Val Leu Arg Ser Ile Arg Cys Leu Arg Asn Val Ile His Trp Asn160 165 170 ctc atc acc acc ttc att ctg aga aac atc gcg tgg ttc ctg ctgcaa 698 Leu Ile Thr Thr Phe Ile Leu Arg Asn Ile Ala Trp Phe Leu Leu Gln175 180 185 ctc atc gac cac gaa gtg cac gag ggc aat gag gtc tgg tgc cgctgc 746 Leu Ile Asp His Glu Val His Glu Gly Asn Glu Val Trp Cys Arg Cys190 195 200 205 atc acc acc atc ttc aac tat ttt gtg gtc acc aac ttc ttctgg atg 794 Ile Thr Thr Ile Phe Asn Tyr Phe Val Val Thr Asn Phe Phe TrpMet 210 215 220 ttt gtg gag ggc tgc tac ctg cac acg gcc att gtc atg acgtac tcc 842 Phe Val Glu Gly Cys Tyr Leu His Thr Ala Ile Val Met Thr TyrSer 225 230 235 aca gag cac ctg cgc aag tgg ctt ttc ctc ttc att gga tggtgc att 890 Thr Glu His Leu Arg Lys Trp Leu Phe Leu Phe Ile Gly Trp CysIle 240 245 250 ccc tgc cct atc atc atc gcc tgg gca gtt ggc aaa ctc tactat gag 938 Pro Cys Pro Ile Ile Ile Ala Trp Ala Val Gly Lys Leu Tyr TyrGlu 255 260 265 aat gag cag tgc tgg ttt ggc aag gaa gct ggt gat ttg gtggac tac 986 Asn Glu Gln Cys Trp Phe Gly Lys Glu Ala Gly Asp Leu Val AspTyr 270 275 280 285 atc tac cag ggc ccc gtc atg ctt gtg ctg ttg atc aatttt gta ttt 1034 Ile Tyr Gln Gly Pro Val Met Leu Val Leu Leu Ile Asn PheVal Phe 290 295 300 ctg ttt aac atc gtc agg atc ctg atg acg aag tta cgagca tcc acc 1082 Leu Phe Asn Ile Val Arg Ile Leu Met Thr Lys Leu Arg AlaSer Thr 305 310 315 acg tcc gag aca atc caa tac agg aag gca gtg aag gccacg ctg gtc 1130 Thr Ser Glu Thr Ile Gln Tyr Arg Lys Ala Val Lys Ala ThrLeu Val 320 325 330 ctc ctc ccc ctg ttg ggc atc acc tac atg ctc ttc tttgtc aat cct 1178 Leu Leu Pro Leu Leu Gly Ile Thr Tyr Met Leu Phe Phe ValAsn Pro 335 340 345 ggc gag gac gac ctg tcc cag att gtg ttc atc tac ttcaac tct ttc 1226 Gly Glu Asp Asp Leu Ser Gln Ile Val Phe Ile Tyr Phe AsnSer Phe 350 355 360 365 ctg cag tcc ttc cag ggt ttc ttt gtg tcc gtt ttctac tgc ttc ttc 1274 Leu Gln Ser Phe Gln Gly Phe Phe Val Ser Val Phe TyrCys Phe Phe 370 375 380 aat gga gag gtg cgc gcg gcc ctg aga aac ggg tggcac cgc tgg cag 1322 Asn Gly Glu Val Arg Ala Ala Leu Arg Asn Gly Trp HisArg Trp Gln 385 390 395 gac cac cac gcc ctc cgg gtg cct gtg gcc cgg gccatg tcc atc cct 1370 Asp His His Ala Leu Arg Val Pro Val Ala Arg Ala MetSer Ile Pro 400 405 410 acg tcg ccc acc agg atc agc ttc cac agc atc aagcag aca gct gct 1418 Thr Ser Pro Thr Arg Ile Ser Phe His Ser Ile Lys GlnThr Ala Ala 415 420 425 gtg tga ccctctgtca ccgtctgccc ggcagtccaccactgaggca gcttctccat 1474 Val 430 cctttacagc cttcccctgg gtcctccttgctaccctgac ccacagggta caaggtacag 1534 gagaagggag gagaacgaac actcccgcctggaaggaaag gaaagctatg acatgggggg 1594 gctctgaagg accagggccc agtgcagccagccacacatc tccaagcacg aaggagcagg 1654 aggacatcac aggaccctca gaagggatgcatctcacacc atcaagcctc tgtgcaccca 1714 gcctcttttg tggggtcctc actgcagcaccatttacatc tgaagaaact gaggctcaga 1774 gcaggcaggg acctggccaa gtcacatagctacttgcccc acccacagca cccacagttg 1834 gctctgctcc ttgctttcca tctccacacgtgagggcgcc ctctaaaggt gagggagaca 1894 agaatgacct tatctggctt catcccagaagctgtcgagc agagatgacc agccctttac 1954 caaggtagcc ttcttcttcc ccagtctgtttcccatgtgt ctccaggaga atgctggctt 2014 tcagtcggcc atccctcctg ggagtccccaattcagtctg ggctcagtct ggggacctag 2074 accacgggaa gtgagttaga tggaaagtcacactctccac agtgccagac agaagggaga 2134 acagaagcgc ctggggaaga agggtgaggatcccccaaat cagagtatgc ctgggagtga 2194 ttgaaacaag ggccccagga tctcagtgacatcagccagg catctgtgga gttggccaca 2254 attcaagcaa cgagatgttg gagagatattgtgagccagt aataaaggca gaatgtctgc 2314 aggacatatc catgcccctc ttcttactggctaggcccaa gcaggccttc ctgtggagtc 2374 tttaggttca aagggcccga atcattcctgtcaccccaaa gggtggcatc tgcaccaccc 2434 ccagcgtaga ccccacctgt gccagggactaatattctgg aattgggagg gagaggaggc 2494 aaggcccttc aggctccgaa agcaagaagacacagtttga tttcaggctt ctcttccatt 2554 cctctgtccc tggagcagaa gaggggtgttggggcaagcc aacagacttg aaaaggcccc 2614 cgg 2617 26 430 PRT Mus musculus26 Met Gly Thr Pro Gly Ser Leu Pro Ser Ala Gln Leu Leu Leu Cys Leu 1 510 15 Phe Ser Leu Leu Pro Val Leu Gln Val Ala Gln Pro Gly Gln Ala Pro 2025 30 Gln Asp Gln Pro Leu Trp Thr Leu Leu Glu Gln Tyr Cys His Arg Thr 3540 45 Thr Ile Gly Asn Phe Ser Gly Pro Tyr Thr Tyr Cys Asn Thr Thr Leu 5055 60 Asp Gln Ile Gly Thr Cys Trp Pro Gln Ser Ala Pro Gly Ala Leu Val 6570 75 80 Glu Arg Pro Cys Pro Glu Tyr Phe Asn Gly Ile Lys Tyr Asn Thr Thr85 90 95 Arg Asn Ala Tyr Arg Glu Cys Leu Glu Asn Gly Thr Trp Ala Ser Arg100 105 110 Val Asn Tyr Ser His Cys Glu Pro Ile Leu Asp Asp Lys Arg LysTyr 115 120 125 Asp Leu His Tyr Arg Ile Ala Leu Ile Val Asn Tyr Leu GlyHis Cys 130 135 140 Val Ser Val Val Ala Leu Val Ala Ala Phe Leu Leu PheLeu Val Leu 145 150 155 160 Arg Ser Ile Arg Cys Leu Arg Asn Val Ile HisTrp Asn Leu Ile Thr 165 170 175 Thr Phe Ile Leu Arg Asn Ile Ala Trp PheLeu Leu Gln Leu Ile Asp 180 185 190 His Glu Val His Glu Gly Asn Glu ValTrp Cys Arg Cys Ile Thr Thr 195 200 205 Ile Phe Asn Tyr Phe Val Val ThrAsn Phe Phe Trp Met Phe Val Glu 210 215 220 Gly Cys Tyr Leu His Thr AlaIle Val Met Thr Tyr Ser Thr Glu His 225 230 235 240 Leu Arg Lys Trp LeuPhe Leu Phe Ile Gly Trp Cys Ile Pro Cys Pro 245 250 255 Ile Ile Ile AlaTrp Ala Val Gly Lys Leu Tyr Tyr Glu Asn Glu Gln 260 265 270 Cys Trp PheGly Lys Glu Ala Gly Asp Leu Val Asp Tyr Ile Tyr Gln 275 280 285 Gly ProVal Met Leu Val Leu Leu Ile Asn Phe Val Phe Leu Phe Asn 290 295 300 IleVal Arg Ile Leu Met Thr Lys Leu Arg Ala Ser Thr Thr Ser Glu 305 310 315320 Thr Ile Gln Tyr Arg Lys Ala Val Lys Ala Thr Leu Val Leu Leu Pro 325330 335 Leu Leu Gly Ile Thr Tyr Met Leu Phe Phe Val Asn Pro Gly Glu Asp340 345 350 Asp Leu Ser Gln Ile Val Phe Ile Tyr Phe Asn Ser Phe Leu GlnSer 355 360 365 Phe Gln Gly Phe Phe Val Ser Val Phe Tyr Cys Phe Phe AsnGly Glu 370 375 380 Val Arg Ala Ala Leu Arg Asn Gly Trp His Arg Trp GlnAsp His His 385 390 395 400 Ala Leu Arg Val Pro Val Ala Arg Ala Met SerIle Pro Thr Ser Pro 405 410 415 Thr Arg Ile Ser Phe His Ser Ile Lys GlnThr Ala Ala Val 420 425 430 27 1248 DNA Ovis aries CDS (1)..(1248) 27atg gga cgg cgc ccg cag ctc cgg ctt gtc aag gcc ctt ctc ctc ctg 48 MetGly Arg Arg Pro Gln Leu Arg Leu Val Lys Ala Leu Leu Leu Leu 1 5 10 15ggg ctg aac tcc atc tcc gcc tcc ctc cag gac cag cat tgc gag agc 96 GlyLeu Asn Ser Ile Ser Ala Ser Leu Gln Asp Gln His Cys Glu Ser 20 25 30 ttgtcc ctg gcc agc aac gtc tct gga ctg cag tgc aac gct tcc gtg 144 Leu SerLeu Ala Ser Asn Val Ser Gly Leu Gln Cys Asn Ala Ser Val 35 40 45 gac cttaat ggc acc tgc tgg ccc cag agt cct gca ggg cag ttg gtg 192 Asp Leu AsnGly Thr Cys Trp Pro Gln Ser Pro Ala Gly Gln Leu Val 50 55 60 gtt cga ccctgc ctc gta ttt ttc tat ggt gtc cgc tac aat acc aca 240 Val Arg Pro CysLeu Val Phe Phe Tyr Gly Val Arg Tyr Asn Thr Thr 65 70 75 80 agc aat ggctac cgg gtg tgc ctg gcc aat ggc acg tgg gca gcc cgc 288 Ser Asn Gly TyrArg Val Cys Leu Ala Asn Gly Thr Trp Ala Ala Arg 85 90 95 gtg aat cac tccgag tgc caa gag atc ctc agc gaa gga gag aag agc 336 Val Asn His Ser GluCys Gln Glu Ile Leu Ser Glu Gly Glu Lys Ser 100 105 110 aag gcg cac taccac atc gcc gtc atc atc aac tac ctg ggc cac tgc 384 Lys Ala His Tyr HisIle Ala Val Ile Ile Asn Tyr Leu Gly His Cys 115 120 125 atc tcc ctg gcggcc ctc ctg gtg gcc ttt gtc ctc ttt ctg cgg ctc 432 Ile Ser Leu Ala AlaLeu Leu Val Ala Phe Val Leu Phe Leu Arg Leu 130 135 140 agg agc atc cggtgc gtg aga aac atc atc cac tgg aac ctc atc tca 480 Arg Ser Ile Arg CysVal Arg Asn Ile Ile His Trp Asn Leu Ile Ser 145 150 155 160 gcc ttc atcctg cgc aat gcc acg tgg ttc gtg gtc cag ctc acc atg 528 Ala Phe Ile LeuArg Asn Ala Thr Trp Phe Val Val Gln Leu Thr Met 165 170 175 agc ccc gaagtc cat cag agc aac gtg ggc tgg tgc agg ctg gtg aca 576 Ser Pro Glu ValHis Gln Ser Asn Val Gly Trp Cys Arg Leu Val Thr 180 185 190 gcc gcc tacaac tac ttc cac gtg acc aac ttc ttc tgg atg ttc ggc 624 Ala Ala Tyr AsnTyr Phe His Val Thr Asn Phe Phe Trp Met Phe Gly 195 200 205 gag ggc tgctac ctg cac acg gct gtc gtg ctc aca tac tcc acg gac 672 Glu Gly Cys TyrLeu His Thr Ala Val Val Leu Thr Tyr Ser Thr Asp 210 215 220 cgg ctg cgcaaa tgg atg ttt atc tgc atc ggc tgg ggt gtg ccc ttc 720 Arg Leu Arg LysTrp Met Phe Ile Cys Ile Gly Trp Gly Val Pro Phe 225 230 235 240 ccc atcatt gtg gcc tgg gcc att gga aag ttg tac tac gac aat gag 768 Pro Ile IleVal Ala Trp Ala Ile Gly Lys Leu Tyr Tyr Asp Asn Glu 245 250 255 aag tgctgg ttt ggc aaa agg cct ggg gtg tac act gat tac atc tac 816 Lys Cys TrpPhe Gly Lys Arg Pro Gly Val Tyr Thr Asp Tyr Ile Tyr 260 265 270 cag ggcccg atg atc ttg gtc ctg ctg atc aat ttc atc ttc ctt ttc 864 Gln Gly ProMet Ile Leu Val Leu Leu Ile Asn Phe Ile Phe Leu Phe 275 280 285 aac atcgtt cgc atc ctc atg acc aaa ctc cgg gca tcc acc acc tct 912 Asn Ile ValArg Ile Leu Met Thr Lys Leu Arg Ala Ser Thr Thr Ser 290 295 300 gag accatt cag tac agg aag gct gtg aag gcc act ctg gtg ctg ctc 960 Glu Thr IleGln Tyr Arg Lys Ala Val Lys Ala Thr Leu Val Leu Leu 305 310 315 320 cccctc ctg ggc atc acg tac atg ctg ttc ttc gtg aac ccc ggg gag 1008 Pro LeuLeu Gly Ile Thr Tyr Met Leu Phe Phe Val Asn Pro Gly Glu 325 330 335 gacgag gtc tcc cgg gtc gtc ttc atc tac ttc aac tcc ttc ctg gaa 1056 Asp GluVal Ser Arg Val Val Phe Ile Tyr Phe Asn Ser Phe Leu Glu 340 345 350 tctttc cag ggc ttc ttc gtg tct gtg ttc tac tgc ttc ctc aac agc 1104 Ser PheGln Gly Phe Phe Val Ser Val Phe Tyr Cys Phe Leu Asn Ser 355 360 365 gaggtc cgc tct gcc atc cgg aag agg tgg cac cgc tgg cag gac aag 1152 Glu ValArg Ser Ala Ile Arg Lys Arg Trp His Arg Trp Gln Asp Lys 370 375 380 cactca atc cgt gcc cgt gtg gct cgc gcc atg tcc atc ccc acc tcc 1200 His SerIle Arg Ala Arg Val Ala Arg Ala Met Ser Ile Pro Thr Ser 385 390 395 400ccc acc cgt gtc agc ttt cac agc atc aag cag tcc aca gca gtg tga 1248 ProThr Arg Val Ser Phe His Ser Ile Lys Gln Ser Thr Ala Val 405 410 415 28415 PRT Ovis aries 28 Met Gly Arg Arg Pro Gln Leu Arg Leu Val Lys AlaLeu Leu Leu Leu 1 5 10 15 Gly Leu Asn Ser Ile Ser Ala Ser Leu Gln AspGln His Cys Glu Ser 20 25 30 Leu Ser Leu Ala Ser Asn Val Ser Gly Leu GlnCys Asn Ala Ser Val 35 40 45 Asp Leu Asn Gly Thr Cys Trp Pro Gln Ser ProAla Gly Gln Leu Val 50 55 60 Val Arg Pro Cys Leu Val Phe Phe Tyr Gly ValArg Tyr Asn Thr Thr 65 70 75 80 Ser Asn Gly Tyr Arg Val Cys Leu Ala AsnGly Thr Trp Ala Ala Arg 85 90 95 Val Asn His Ser Glu Cys Gln Glu Ile LeuSer Glu Gly Glu Lys Ser 100 105 110 Lys Ala His Tyr His Ile Ala Val IleIle Asn Tyr Leu Gly His Cys 115 120 125 Ile Ser Leu Ala Ala Leu Leu ValAla Phe Val Leu Phe Leu Arg Leu 130 135 140 Arg Ser Ile Arg Cys Val ArgAsn Ile Ile His Trp Asn Leu Ile Ser 145 150 155 160 Ala Phe Ile Leu ArgAsn Ala Thr Trp Phe Val Val Gln Leu Thr Met 165 170 175 Ser Pro Glu ValHis Gln Ser Asn Val Gly Trp Cys Arg Leu Val Thr 180 185 190 Ala Ala TyrAsn Tyr Phe His Val Thr Asn Phe Phe Trp Met Phe Gly 195 200 205 Glu GlyCys Tyr Leu His Thr Ala Val Val Leu Thr Tyr Ser Thr Asp 210 215 220 ArgLeu Arg Lys Trp Met Phe Ile Cys Ile Gly Trp Gly Val Pro Phe 225 230 235240 Pro Ile Ile Val Ala Trp Ala Ile Gly Lys Leu Tyr Tyr Asp Asn Glu 245250 255 Lys Cys Trp Phe Gly Lys Arg Pro Gly Val Tyr Thr Asp Tyr Ile Tyr260 265 270 Gln Gly Pro Met Ile Leu Val Leu Leu Ile Asn Phe Ile Phe LeuPhe 275 280 285 Asn Ile Val Arg Ile Leu Met Thr Lys Leu Arg Ala Ser ThrThr Ser 290 295 300 Glu Thr Ile Gln Tyr Arg Lys Ala Val Lys Ala Thr LeuVal Leu Leu 305 310 315 320 Pro Leu Leu Gly Ile Thr Tyr Met Leu Phe PheVal Asn Pro Gly Glu 325 330 335 Asp Glu Val Ser Arg Val Val Phe Ile TyrPhe Asn Ser Phe Leu Glu 340 345 350 Ser Phe Gln Gly Phe Phe Val Ser ValPhe Tyr Cys Phe Leu Asn Ser 355 360 365 Glu Val Arg Ser Ala Ile Arg LysArg Trp His Arg Trp Gln Asp Lys 370 375 380 His Ser Ile Arg Ala Arg ValAla Arg Ala Met Ser Ile Pro Thr Ser 385 390 395 400 Pro Thr Arg Val SerPhe His Ser Ile Lys Gln Ser Thr Ala Val 405 410 415 29 1248 DNA Xenopuslaevis CDS (1)..(1248) 29 atg ctg ttg gcc aaa act cca tgt cta cta ctggtg cag gtg atc gct 48 Met Leu Leu Ala Lys Thr Pro Cys Leu Leu Leu ValGln Val Ile Ala 1 5 10 15 gct gga atc agt ttt gcc ctc acc tct ctc caggac caa tgt gaa acc 96 Ala Gly Ile Ser Phe Ala Leu Thr Ser Leu Gln AspGln Cys Glu Thr 20 25 30 ctg cag cac aat tct aac ttc aca ggt ctt gcc tgcaac gct tcc att 144 Leu Gln His Asn Ser Asn Phe Thr Gly Leu Ala Cys AsnAla Ser Ile 35 40 45 gat atg atc ggc act tgc tgg ccc agt act gca gct ggacag atg gtg 192 Asp Met Ile Gly Thr Cys Trp Pro Ser Thr Ala Ala Gly GlnMet Val 50 55 60 gcc aga ccc tgc ccc gag tac ttc cat ggg gtg caa tac aacaca aca 240 Ala Arg Pro Cys Pro Glu Tyr Phe His Gly Val Gln Tyr Asn ThrThr 65 70 75 80 ggg aat gtg tac aga gaa tgt cac ctg aac ggc agc tgg gctggg aga 288 Gly Asn Val Tyr Arg Glu Cys His Leu Asn Gly Ser Trp Ala GlyArg 85 90 95 gga gac tac gct caa tgc cag gag att cta aag caa gag aag aaaacc 336 Gly Asp Tyr Ala Gln Cys Gln Glu Ile Leu Lys Gln Glu Lys Lys Thr100 105 110 aaa gtt cat tat cac ata gcc atc gtg att aac ttc ctg ggt cactcc 384 Lys Val His Tyr His Ile Ala Ile Val Ile Asn Phe Leu Gly His Ser115 120 125 att tcc ctt tgt gct ctc ctg gtg gct ttt atc ctg ttc ttg aggttg 432 Ile Ser Leu Cys Ala Leu Leu Val Ala Phe Ile Leu Phe Leu Arg Leu130 135 140 agg agc atc cgg tgc cta cgt aat atc atc cac tgg aac ctg atcacg 480 Arg Ser Ile Arg Cys Leu Arg Asn Ile Ile His Trp Asn Leu Ile Thr145 150 155 160 gct ttt att ctg cgt aat gta acc tgg ttt gtg atg cag ctcact ctc 528 Ala Phe Ile Leu Arg Asn Val Thr Trp Phe Val Met Gln Leu ThrLeu 165 170 175 agc cat gaa gcc cac gac agc aat gtg gtt tgg tgc cgc ctggtc acc 576 Ser His Glu Ala His Asp Ser Asn Val Val Trp Cys Arg Leu ValThr 180 185 190 atc gct cac aat tat ttt tat gtt acc aac ttc ttc tgg atgttt ggg 624 Ile Ala His Asn Tyr Phe Tyr Val Thr Asn Phe Phe Trp Met PheGly 195 200 205 gag ggc tgt tac ctg cac acg gcc att gtt cta acc tac tcaact gac 672 Glu Gly Cys Tyr Leu His Thr Ala Ile Val Leu Thr Tyr Ser ThrAsp 210 215 220 aaa ctg cgc aaa tgg atg ttc atc tgt att ggc tgg tgt atcccc ttt 720 Lys Leu Arg Lys Trp Met Phe Ile Cys Ile Gly Trp Cys Ile ProPhe 225 230 235 240 ccc atc att gtg gct tgg gcc att ggc aaa ctt tac tacgac aat gaa 768 Pro Ile Ile Val Ala Trp Ala Ile Gly Lys Leu Tyr Tyr AspAsn Glu 245 250 255 aag tgc tgg ttt ggg aag aaa gcg gga gtc tac aca gatttt atc tac 816 Lys Cys Trp Phe Gly Lys Lys Ala Gly Val Tyr Thr Asp PheIle Tyr 260 265 270 caa gga cct gtc atc ctt gtg ctg ctg atc aac ttc atattt tta ttc 864 Gln Gly Pro Val Ile Leu Val Leu Leu Ile Asn Phe Ile PheLeu Phe 275 280 285 aac att gta cgg att ctg atg aca aag ctc aga gct tccacc act tca 912 Asn Ile Val Arg Ile Leu Met Thr Lys Leu Arg Ala Ser ThrThr Ser 290 295 300 gag acc ata cag tac agg aaa gct gtt aaa gcc acc ctggtg ctc ctg 960 Glu Thr Ile Gln Tyr Arg Lys Ala Val Lys Ala Thr Leu ValLeu Leu 305 310 315 320 cct ttg ctt ggg atc acc tac atg ctt ttc ttt gtgacg ccc ggg gag 1008 Pro Leu Leu Gly Ile Thr Tyr Met Leu Phe Phe Val ThrPro Gly Glu 325 330 335 gat gaa atc tca cgt atc gtc ttt atc tat ttc aactct ttc ctg cag 1056 Asp Glu Ile Ser Arg Ile Val Phe Ile Tyr Phe Asn SerPhe Leu Gln 340 345 350 tcc ttt cag ggt ttc ttt gtt tca gtt ttc tac tgcttc ctt aat agt 1104 Ser Phe Gln Gly Phe Phe Val Ser Val Phe Tyr Cys PheLeu Asn Ser 355 360 365 gag gtg cgc tca gca gtc cgg aag cga tgg cac cgatgg cag gac aag 1152 Glu Val Arg Ser Ala Val Arg Lys Arg Trp His Arg TrpGln Asp Lys 370 375 380 cat tca atc cgt gct cgc gtg gcc cgt gcc atg tccatt ccc aca tca 1200 His Ser Ile Arg Ala Arg Val Ala Arg Ala Met Ser IlePro Thr Ser 385 390 395 400 ccc act cgg att agt ttc cac agc atc aag caatct tct gcc att tga 1248 Pro Thr Arg Ile Ser Phe His Ser Ile Lys Gln SerSer Ala Ile 405 410 415 30 415 PRT Xenopus laevis 30 Met Leu Leu Ala LysThr Pro Cys Leu Leu Leu Val Gln Val Ile Ala 1 5 10 15 Ala Gly Ile SerPhe Ala Leu Thr Ser Leu Gln Asp Gln Cys Glu Thr 20 25 30 Leu Gln His AsnSer Asn Phe Thr Gly Leu Ala Cys Asn Ala Ser Ile 35 40 45 Asp Met Ile GlyThr Cys Trp Pro Ser Thr Ala Ala Gly Gln Met Val 50 55 60 Ala Arg Pro CysPro Glu Tyr Phe His Gly Val Gln Tyr Asn Thr Thr 65 70 75 80 Gly Asn ValTyr Arg Glu Cys His Leu Asn Gly Ser Trp Ala Gly Arg 85 90 95 Gly Asp TyrAla Gln Cys Gln Glu Ile Leu Lys Gln Glu Lys Lys Thr 100 105 110 Lys ValHis Tyr His Ile Ala Ile Val Ile Asn Phe Leu Gly His Ser 115 120 125 IleSer Leu Cys Ala Leu Leu Val Ala Phe Ile Leu Phe Leu Arg Leu 130 135 140Arg Ser Ile Arg Cys Leu Arg Asn Ile Ile His Trp Asn Leu Ile Thr 145 150155 160 Ala Phe Ile Leu Arg Asn Val Thr Trp Phe Val Met Gln Leu Thr Leu165 170 175 Ser His Glu Ala His Asp Ser Asn Val Val Trp Cys Arg Leu ValThr 180 185 190 Ile Ala His Asn Tyr Phe Tyr Val Thr Asn Phe Phe Trp MetPhe Gly 195 200 205 Glu Gly Cys Tyr Leu His Thr Ala Ile Val Leu Thr TyrSer Thr Asp 210 215 220 Lys Leu Arg Lys Trp Met Phe Ile Cys Ile Gly TrpCys Ile Pro Phe 225 230 235 240 Pro Ile Ile Val Ala Trp Ala Ile Gly LysLeu Tyr Tyr Asp Asn Glu 245 250 255 Lys Cys Trp Phe Gly Lys Lys Ala GlyVal Tyr Thr Asp Phe Ile Tyr 260 265 270 Gln Gly Pro Val Ile Leu Val LeuLeu Ile Asn Phe Ile Phe Leu Phe 275 280 285 Asn Ile Val Arg Ile Leu MetThr Lys Leu Arg Ala Ser Thr Thr Ser 290 295 300 Glu Thr Ile Gln Tyr ArgLys Ala Val Lys Ala Thr Leu Val Leu Leu 305 310 315 320 Pro Leu Leu GlyIle Thr Tyr Met Leu Phe Phe Val Thr Pro Gly Glu 325 330 335 Asp Glu IleSer Arg Ile Val Phe Ile Tyr Phe Asn Ser Phe Leu Gln 340 345 350 Ser PheGln Gly Phe Phe Val Ser Val Phe Tyr Cys Phe Leu Asn Ser 355 360 365 GluVal Arg Ser Ala Val Arg Lys Arg Trp His Arg Trp Gln Asp Lys 370 375 380His Ser Ile Arg Ala Arg Val Ala Arg Ala Met Ser Ile Pro Thr Ser 385 390395 400 Pro Thr Arg Ile Ser Phe His Ser Ile Lys Gln Ser Ser Ala Ile 405410 415 31 1242 DNA Xenopus laevis CDS (1)..(1242) 31 atg gac agc accatc ttt gag att atc att gat gaa ttt gat gcc aac 48 Met Asp Ser Thr IlePhe Glu Ile Ile Ile Asp Glu Phe Asp Ala Asn 1 5 10 15 tgc agc ctt ttggat gct ttt cag gac agt ttt ttg cac tct gag tcc 96 Cys Ser Leu Leu AspAla Phe Gln Asp Ser Phe Leu His Ser Glu Ser 20 25 30 tcc tct ttc ttt ggcttt gaa ggt ccc tat tgt agc gct acc att gac 144 Ser Ser Phe Phe Gly PheGlu Gly Pro Tyr Cys Ser Ala Thr Ile Asp 35 40 45 cag att ggc acg tgc tggccc agg agc cta gcc ggg gaa ctt gtg gaa 192 Gln Ile Gly Thr Cys Trp ProArg Ser Leu Ala Gly Glu Leu Val Glu 50 55 60 aga ccc tgc ccg gat tcc ttcaat ggg atc aga tac aac aca act aga 240 Arg Pro Cys Pro Asp Ser Phe AsnGly Ile Arg Tyr Asn Thr Thr Arg 65 70 75 80 aac gtc tac aga gaa tgc tttgag aat gga acc tgg gcg tcc tgg atg 288 Asn Val Tyr Arg Glu Cys Phe GluAsn Gly Thr Trp Ala Ser Trp Met 85 90 95 aat tac tct cag tgt gtg ccc attctg gat aat aag agg aag tac gcc 336 Asn Tyr Ser Gln Cys Val Pro Ile LeuAsp Asn Lys Arg Lys Tyr Ala 100 105 110 ctt cat tac aag att gct ctc atcata aac tac ctg ggg cac tgc atc 384 Leu His Tyr Lys Ile Ala Leu Ile IleAsn Tyr Leu Gly His Cys Ile 115 120 125 tcc atc ttg gct ctc gtt atc gctttc ttg ctc ttt ctg tgt ttg agg 432 Ser Ile Leu Ala Leu Val Ile Ala PheLeu Leu Phe Leu Cys Leu Arg 130 135 140 agt ata aga tgc ctt cgg aac attatc cac tgg aat tta atc act act 480 Ser Ile Arg Cys Leu Arg Asn Ile IleHis Trp Asn Leu Ile Thr Thr 145 150 155 160 ttc atc ctg agg aac atc atgtgg ttc ctg ctg cag atg att gac cat 528 Phe Ile Leu Arg Asn Ile Met TrpPhe Leu Leu Gln Met Ile Asp His 165 170 175 aac att cat gaa agc aac gaggtc tgg tgt cgg tgt atc aca act att 576 Asn Ile His Glu Ser Asn Glu ValTrp Cys Arg Cys Ile Thr Thr Ile 180 185 190 tac aat tac ttt gtg gtg accaac ttc ttc tgg atg ttt gtg gaa gga 624 Tyr Asn Tyr Phe Val Val Thr AsnPhe Phe Trp Met Phe Val Glu Gly 195 200 205 tgt tac cta cac aca gct atagtg atg aca tac tca acg gac aaa ctt 672 Cys Tyr Leu His Thr Ala Ile ValMet Thr Tyr Ser Thr Asp Lys Leu 210 215 220 agg aaa tgg gtg ttc ctc ttcata gga tgg tgt att cca tct ccg atc 720 Arg Lys Trp Val Phe Leu Phe IleGly Trp Cys Ile Pro Ser Pro Ile 225 230 235 240 att gtc acc tgg gcc atctgc aag ctt ttc tat gaa aat gaa cag tgt 768 Ile Val Thr Trp Ala Ile CysLys Leu Phe Tyr Glu Asn Glu Gln Cys 245 250 255 tgg att ggg aag gag cccggg aaa tac att gat tac att tac cag ggc 816 Trp Ile Gly Lys Glu Pro GlyLys Tyr Ile Asp Tyr Ile Tyr Gln Gly 260 265 270 cgg gtg att ctc gta cttctg ata aat ttt gtg ttc tta ttc aac att 864 Arg Val Ile Leu Val Leu LeuIle Asn Phe Val Phe Leu Phe Asn Ile 275 280 285 gta aga att ttg atg acaaaa ctg aga gct tca act aca tct gaa acg 912 Val Arg Ile Leu Met Thr LysLeu Arg Ala Ser Thr Thr Ser Glu Thr 290 295 300 ata cag tac agg aag gctgtg aag gca acg tta gtc ctt ctc cct ctt 960 Ile Gln Tyr Arg Lys Ala ValLys Ala Thr Leu Val Leu Leu Pro Leu 305 310 315 320 ctg gga atc acc tacatg ctc ttc ttc gtc aac cct gga gag gat gac 1008 Leu Gly Ile Thr Tyr MetLeu Phe Phe Val Asn Pro Gly Glu Asp Asp 325 330 335 gtt tct cag atc gttttt att tac ttc aac tcg ttt ctt cag tcc ttt 1056 Val Ser Gln Ile Val PheIle Tyr Phe Asn Ser Phe Leu Gln Ser Phe 340 345 350 cag ggt ttc ttt gtgtca gta ttt tac tgc ttc ctt aat ggg gag gtc 1104 Gln Gly Phe Phe Val SerVal Phe Tyr Cys Phe Leu Asn Gly Glu Val 355 360 365 cgg tcg gct gca aggaaa aga tgg cac cgc tgg caa gac cac cat tct 1152 Arg Ser Ala Ala Arg LysArg Trp His Arg Trp Gln Asp His His Ser 370 375 380 ctg cgg gtt cgg gtagcc aga gcc atg tcc ata cca aca tca ccg acc 1200 Leu Arg Val Arg Val AlaArg Ala Met Ser Ile Pro Thr Ser Pro Thr 385 390 395 400 aga atc agc tttcac agt ata aag caa acg gca gcc gtc tga 1242 Arg Ile Ser Phe His Ser IleLys Gln Thr Ala Ala Val 405 410 32 413 PRT Xenopus laevis 32 Met Asp SerThr Ile Phe Glu Ile Ile Ile Asp Glu Phe Asp Ala Asn 1 5 10 15 Cys SerLeu Leu Asp Ala Phe Gln Asp Ser Phe Leu His Ser Glu Ser 20 25 30 Ser SerPhe Phe Gly Phe Glu Gly Pro Tyr Cys Ser Ala Thr Ile Asp 35 40 45 Gln IleGly Thr Cys Trp Pro Arg Ser Leu Ala Gly Glu Leu Val Glu 50 55 60 Arg ProCys Pro Asp Ser Phe Asn Gly Ile Arg Tyr Asn Thr Thr Arg 65 70 75 80 AsnVal Tyr Arg Glu Cys Phe Glu Asn Gly Thr Trp Ala Ser Trp Met 85 90 95 AsnTyr Ser Gln Cys Val Pro Ile Leu Asp Asn Lys Arg Lys Tyr Ala 100 105 110Leu His Tyr Lys Ile Ala Leu Ile Ile Asn Tyr Leu Gly His Cys Ile 115 120125 Ser Ile Leu Ala Leu Val Ile Ala Phe Leu Leu Phe Leu Cys Leu Arg 130135 140 Ser Ile Arg Cys Leu Arg Asn Ile Ile His Trp Asn Leu Ile Thr Thr145 150 155 160 Phe Ile Leu Arg Asn Ile Met Trp Phe Leu Leu Gln Met IleAsp His 165 170 175 Asn Ile His Glu Ser Asn Glu Val Trp Cys Arg Cys IleThr Thr Ile 180 185 190 Tyr Asn Tyr Phe Val Val Thr Asn Phe Phe Trp MetPhe Val Glu Gly 195 200 205 Cys Tyr Leu His Thr Ala Ile Val Met Thr TyrSer Thr Asp Lys Leu 210 215 220 Arg Lys Trp Val Phe Leu Phe Ile Gly TrpCys Ile Pro Ser Pro Ile 225 230 235 240 Ile Val Thr Trp Ala Ile Cys LysLeu Phe Tyr Glu Asn Glu Gln Cys 245 250 255 Trp Ile Gly Lys Glu Pro GlyLys Tyr Ile Asp Tyr Ile Tyr Gln Gly 260 265 270 Arg Val Ile Leu Val LeuLeu Ile Asn Phe Val Phe Leu Phe Asn Ile 275 280 285 Val Arg Ile Leu MetThr Lys Leu Arg Ala Ser Thr Thr Ser Glu Thr 290 295 300 Ile Gln Tyr ArgLys Ala Val Lys Ala Thr Leu Val Leu Leu Pro Leu 305 310 315 320 Leu GlyIle Thr Tyr Met Leu Phe Phe Val Asn Pro Gly Glu Asp Asp 325 330 335 ValSer Gln Ile Val Phe Ile Tyr Phe Asn Ser Phe Leu Gln Ser Phe 340 345 350Gln Gly Phe Phe Val Ser Val Phe Tyr Cys Phe Leu Asn Gly Glu Val 355 360365 Arg Ser Ala Ala Arg Lys Arg Trp His Arg Trp Gln Asp His His Ser 370375 380 Leu Arg Val Arg Val Ala Arg Ala Met Ser Ile Pro Thr Ser Pro Thr385 390 395 400 Arg Ile Ser Phe His Ser Ile Lys Gln Thr Ala Ala Val 405410 33 1338 DNA Ameiurus nebulosus CDS (1)..(1338) 33 atg cat ttc cttcta cgt cct cag gtg ttt tct atc tgg atc aca cta 48 Met His Phe Leu LeuArg Pro Gln Val Phe Ser Ile Trp Ile Thr Leu 1 5 10 15 ttc tct ggg gccaca gct gag ctc aca tgc gac act ctg ctc ctg ctc 96 Phe Ser Gly Ala ThrAla Glu Leu Thr Cys Asp Thr Leu Leu Leu Leu 20 25 30 tcc acc aac cgc acagct cgc aca tta ata cta tgg aac cag acg tcg 144 Ser Thr Asn Arg Thr AlaArg Thr Leu Ile Leu Trp Asn Gln Thr Ser 35 40 45 agc tca agt aat gcc acaggt aca agc tca agt aat gcc aca ggt aca 192 Ser Ser Ser Asn Ala Thr GlyThr Ser Ser Ser Asn Ala Thr Gly Thr 50 55 60 agc tca agc aat gcc aca ggtttg ttc tgt aat ata tct ata gat ggc 240 Ser Ser Ser Asn Ala Thr Gly LeuPhe Cys Asn Ile Ser Ile Asp Gly 65 70 75 80 atc ggg acg tgt tgg ccc aggagc aac gca ggg gaa ata gta tca cgt 288 Ile Gly Thr Cys Trp Pro Arg SerAsn Ala Gly Glu Ile Val Ser Arg 85 90 95 cca tgt cct gag acc ttc ttg ggtgtc cgc tac aac acc acc aat aac 336 Pro Cys Pro Glu Thr Phe Leu Gly ValArg Tyr Asn Thr Thr Asn Asn 100 105 110 gtc tac aga gaa tgc ctc gcc aatgga acg tgg gcg aag aag ggg aat 384 Val Tyr Arg Glu Cys Leu Ala Asn GlyThr Trp Ala Lys Lys Gly Asn 115 120 125 tat tct cag tgt cag gaa att ctcaat gaa gag aaa aag agc aag ctg 432 Tyr Ser Gln Cys Gln Glu Ile Leu AsnGlu Glu Lys Lys Ser Lys Leu 130 135 140 cac tac cac att gca gtg att ataaac tac ctg ggc cac tgc atc tct 480 His Tyr His Ile Ala Val Ile Ile AsnTyr Leu Gly His Cys Ile Ser 145 150 155 160 ctc gga gcc ctg ctg gtt gccttc att ctc ttt atg agg ctg agg atg 528 Leu Gly Ala Leu Leu Val Ala PheIle Leu Phe Met Arg Leu Arg Met 165 170 175 atc cgc tgc ctc agg aac atcatt cac tgg aat ctg att atg gct ttc 576 Ile Arg Cys Leu Arg Asn Ile IleHis Trp Asn Leu Ile Met Ala Phe 180 185 190 atc ctg cgc aat gct aca tggttc gta gtg cag ctg acc atg aac cca 624 Ile Leu Arg Asn Ala Thr Trp PheVal Val Gln Leu Thr Met Asn Pro 195 200 205 gag gtg cat gag agc aat gtgatc tgg tgc agg ctg gtt aca gca gcg 672 Glu Val His Glu Ser Asn Val IleTrp Cys Arg Leu Val Thr Ala Ala 210 215 220 tat aat tac ttt cat gtg accaac ttc ttc tgg atg ttt ggt gag ggc 720 Tyr Asn Tyr Phe His Val Thr AsnPhe Phe Trp Met Phe Gly Glu Gly 225 230 235 240 tgc tat ctg cac acg gccatc gtg ctg act tac tcc act gat aag ctc 768 Cys Tyr Leu His Thr Ala IleVal Leu Thr Tyr Ser Thr Asp Lys Leu 245 250 255 agg aag tgg ctg ttc atctgt atc ggc tgg tgt att ccc ttt cct atc 816 Arg Lys Trp Leu Phe Ile CysIle Gly Trp Cys Ile Pro Phe Pro Ile 260 265 270 atc gtt gca tgg gcc attggt aag ctg tat tac gac aat gaa aag tgc 864 Ile Val Ala Trp Ala Ile GlyLys Leu Tyr Tyr Asp Asn Glu Lys Cys 275 280 285 tgg ttt gga aaa cga gctggt gtt tat act gac tac atc tat cag ggc 912 Trp Phe Gly Lys Arg Ala GlyVal Tyr Thr Asp Tyr Ile Tyr Gln Gly 290 295 300 ccc atg atc ctt gtt cttctg atc aac ttt att ttc ctt ttc aac atc 960 Pro Met Ile Leu Val Leu LeuIle Asn Phe Ile Phe Leu Phe Asn Ile 305 310 315 320 gtg agg atc ctg atgaca aag cta aga gcc tcc acc aca tca gag acg 1008 Val Arg Ile Leu Met ThrLys Leu Arg Ala Ser Thr Thr Ser Glu Thr 325 330 335 att cag tac agg aaagct gtg aag gcc act ctg gtc ctg ctg cct ctc 1056 Ile Gln Tyr Arg Lys AlaVal Lys Ala Thr Leu Val Leu Leu Pro Leu 340 345 350 ctc ggg atc acc tacatg ctt ttc ttt gtt aac cct gga gag gac gag 1104 Leu Gly Ile Thr Tyr MetLeu Phe Phe Val Asn Pro Gly Glu Asp Glu 355 360 365 atc tcc caa atc gtcttc atc tat ttc aat tct ttc ctc gag tcc ttt 1152 Ile Ser Gln Ile Val PheIle Tyr Phe Asn Ser Phe Leu Glu Ser Phe 370 375 380 caa ggt ttc ttc gtgtct gtg ttt tat tgc ttc ctg aac agt gaa gtc 1200 Gln Gly Phe Phe Val SerVal Phe Tyr Cys Phe Leu Asn Ser Glu Val 385 390 395 400 cgt tcg gct gttcgg aag cgc tgg cac cgc cgg cag gac aag cac tca 1248 Arg Ser Ala Val ArgLys Arg Trp His Arg Arg Gln Asp Lys His Ser 405 410 415 atc cgg gca cgggtg gca cgg gcc atg tcc att ccc acc tcg cct act 1296 Ile Arg Ala Arg ValAla Arg Ala Met Ser Ile Pro Thr Ser Pro Thr 420 425 430 cgg gtc agc ttccac agc atc aag caa tcc tca gca gtg tga 1338 Arg Val Ser Phe His Ser IleLys Gln Ser Ser Ala Val 435 440 445 34 445 PRT Ameiurus nebulosus 34 MetHis Phe Leu Leu Arg Pro Gln Val Phe Ser Ile Trp Ile Thr Leu 1 5 10 15Phe Ser Gly Ala Thr Ala Glu Leu Thr Cys Asp Thr Leu Leu Leu Leu 20 25 30Ser Thr Asn Arg Thr Ala Arg Thr Leu Ile Leu Trp Asn Gln Thr Ser 35 40 45Ser Ser Ser Asn Ala Thr Gly Thr Ser Ser Ser Asn Ala Thr Gly Thr 50 55 60Ser Ser Ser Asn Ala Thr Gly Leu Phe Cys Asn Ile Ser Ile Asp Gly 65 70 7580 Ile Gly Thr Cys Trp Pro Arg Ser Asn Ala Gly Glu Ile Val Ser Arg 85 9095 Pro Cys Pro Glu Thr Phe Leu Gly Val Arg Tyr Asn Thr Thr Asn Asn 100105 110 Val Tyr Arg Glu Cys Leu Ala Asn Gly Thr Trp Ala Lys Lys Gly Asn115 120 125 Tyr Ser Gln Cys Gln Glu Ile Leu Asn Glu Glu Lys Lys Ser LysLeu 130 135 140 His Tyr His Ile Ala Val Ile Ile Asn Tyr Leu Gly His CysIle Ser 145 150 155 160 Leu Gly Ala Leu Leu Val Ala Phe Ile Leu Phe MetArg Leu Arg Met 165 170 175 Ile Arg Cys Leu Arg Asn Ile Ile His Trp AsnLeu Ile Met Ala Phe 180 185 190 Ile Leu Arg Asn Ala Thr Trp Phe Val ValGln Leu Thr Met Asn Pro 195 200 205 Glu Val His Glu Ser Asn Val Ile TrpCys Arg Leu Val Thr Ala Ala 210 215 220 Tyr Asn Tyr Phe His Val Thr AsnPhe Phe Trp Met Phe Gly Glu Gly 225 230 235 240 Cys Tyr Leu His Thr AlaIle Val Leu Thr Tyr Ser Thr Asp Lys Leu 245 250 255 Arg Lys Trp Leu PheIle Cys Ile Gly Trp Cys Ile Pro Phe Pro Ile 260 265 270 Ile Val Ala TrpAla Ile Gly Lys Leu Tyr Tyr Asp Asn Glu Lys Cys 275 280 285 Trp Phe GlyLys Arg Ala Gly Val Tyr Thr Asp Tyr Ile Tyr Gln Gly 290 295 300 Pro MetIle Leu Val Leu Leu Ile Asn Phe Ile Phe Leu Phe Asn Ile 305 310 315 320Val Arg Ile Leu Met Thr Lys Leu Arg Ala Ser Thr Thr Ser Glu Thr 325 330335 Ile Gln Tyr Arg Lys Ala Val Lys Ala Thr Leu Val Leu Leu Pro Leu 340345 350 Leu Gly Ile Thr Tyr Met Leu Phe Phe Val Asn Pro Gly Glu Asp Glu355 360 365 Ile Ser Gln Ile Val Phe Ile Tyr Phe Asn Ser Phe Leu Glu SerPhe 370 375 380 Gln Gly Phe Phe Val Ser Val Phe Tyr Cys Phe Leu Asn SerGlu Val 385 390 395 400 Arg Ser Ala Val Arg Lys Arg Trp His Arg Arg GlnAsp Lys His Ser 405 410 415 Ile Arg Ala Arg Val Ala Arg Ala Met Ser IlePro Thr Ser Pro Thr 420 425 430 Arg Val Ser Phe His Ser Ile Lys Gln SerSer Ala Val 435 440 445 35 1442 DNA Ameiurus nebulosus CDS (102)..(1388)35 cagaaatatg gagatggaga ctgctttaca gtcactcggg tcaaacagga tgttaagctg 60aactgattaa taatcctgcc agctgatcaa ctggaaaggg a atg tgg atc tgc cta 116Met Trp Ile Cys Leu 1 5 atg cta aag gtc ttg tct atc ttg tct ttt gtg gttgtg aag gtg tca 164 Met Leu Lys Val Leu Ser Ile Leu Ser Phe Val Val ValLys Val Ser 10 15 20 gct gat ctt acc tgc gat gcc gtg cta atg ctg gct tctgga aac cac 212 Ala Asp Leu Thr Cys Asp Ala Val Leu Met Leu Ala Ser GlyAsn His 25 30 35 aca ttg tac cat ctg gat gcc gct aat cac tct gac act aataac tcg 260 Thr Leu Tyr His Leu Asp Ala Ala Asn His Ser Asp Thr Asn AsnSer 40 45 50 ggt gtg ttt tgc agc aca gtt atc gat ggc atc ggc acc tgc tggccg 308 Gly Val Phe Cys Ser Thr Val Ile Asp Gly Ile Gly Thr Cys Trp Pro55 60 65 cgc agc gtg gcc ggg gag atg gtg tcg cgt ccg tgt ccg gaa ttc ctc356 Arg Ser Val Ala Gly Glu Met Val Ser Arg Pro Cys Pro Glu Phe Leu 7075 80 85 tac gga gtc cga tac aac acc acc aat aaa atc ttc cgg aaa tgt ctt404 Tyr Gly Val Arg Tyr Asn Thr Thr Asn Lys Ile Phe Arg Lys Cys Leu 9095 100 gct aat gga acc tgg gcg ccc aaa agc aac tac tct cag tgc aag gct452 Ala Asn Gly Thr Trp Ala Pro Lys Ser Asn Tyr Ser Gln Cys Lys Ala 105110 115 att ctc aat gta cag agg aag agc aag ctg cat tat cga atc gct gtc500 Ile Leu Asn Val Gln Arg Lys Ser Lys Leu His Tyr Arg Ile Ala Val 120125 130 atc att aac tac ctg ggt cac tgc ttg tca ctg ttc act ctt ctt atc548 Ile Ile Asn Tyr Leu Gly His Cys Leu Ser Leu Phe Thr Leu Leu Ile 135140 145 gcc ttc ata atc ttc tta cga ctc agg agt att cgc tgt tta agg aac596 Ala Phe Ile Ile Phe Leu Arg Leu Arg Ser Ile Arg Cys Leu Arg Asn 150155 160 165 atc atc cac tgg aat cta acc tct gcc ttc atc ctg aga aat gcgacg 644 Ile Ile His Trp Asn Leu Thr Ser Ala Phe Ile Leu Arg Asn Ala Thr170 175 180 tgg ttc atc gtt cag ctc acc atg aac cct gat gta cac gag agcaac 692 Trp Phe Ile Val Gln Leu Thr Met Asn Pro Asp Val His Glu Ser Asn185 190 195 gtg cca tgg tgc cgt tta gtg acg acg gca tat aac tac ttc cacatg 740 Val Pro Trp Cys Arg Leu Val Thr Thr Ala Tyr Asn Tyr Phe His Met200 205 210 gcc aat ttt ttc tgg atg ttc ggc gaa ggc tgt tat ctt cac acagcc 788 Ala Asn Phe Phe Trp Met Phe Gly Glu Gly Cys Tyr Leu His Thr Ala215 220 225 atc gtg ctc acc tac tcc act gac aaa ctc aag aaa tgg atg ttcatc 836 Ile Val Leu Thr Tyr Ser Thr Asp Lys Leu Lys Lys Trp Met Phe Ile230 235 240 245 tgc atc gga tgg tgt att cct tcg cct att atc gtc gcc tgggcc atc 884 Cys Ile Gly Trp Cys Ile Pro Ser Pro Ile Ile Val Ala Trp AlaIle 250 255 260 gga aag ctg tac tac gac aac gag aag tgt tgg ttt ggg aagaga gca 932 Gly Lys Leu Tyr Tyr Asp Asn Glu Lys Cys Trp Phe Gly Lys ArgAla 265 270 275 ggc ata tac aca gac tac atc tac cag ggc ccc atg atc ctggta ctt 980 Gly Ile Tyr Thr Asp Tyr Ile Tyr Gln Gly Pro Met Ile Leu ValLeu 280 285 290 atg atc aat ttc gtg ttc ctc ttc aac ata gta agg atc ctcatg acc 1028 Met Ile Asn Phe Val Phe Leu Phe Asn Ile Val Arg Ile Leu MetThr 295 300 305 aaa ctc cgg gcc tcc acc aca tcc gaa acc atc cag tac aggaag gcg 1076 Lys Leu Arg Ala Ser Thr Thr Ser Glu Thr Ile Gln Tyr Arg LysAla 310 315 320 325 gtg aag gcc acg ctc gtg tta ctg cct ctg ctc ggg atcacg tac atg 1124 Val Lys Ala Thr Leu Val Leu Leu Pro Leu Leu Gly Ile ThrTyr Met 330 335 340 ctg ttc ttc gta aac cca gga gag gac gaa atc tcg cagatc gtc ttc 1172 Leu Phe Phe Val Asn Pro Gly Glu Asp Glu Ile Ser Gln IleVal Phe 345 350 355 atc tac ttc aat tcc ttt ctg cag tcc ttt cag ggc ttcttt gtg tcc 1220 Ile Tyr Phe Asn Ser Phe Leu Gln Ser Phe Gln Gly Phe PheVal Ser 360 365 370 gtg ttc tac tgt ttt cta aac agc gag gtc cgc tcg gctgtt cgg aag 1268 Val Phe Tyr Cys Phe Leu Asn Ser Glu Val Arg Ser Ala ValArg Lys 375 380 385 cac tgg cac cgc tgg cag gac cac cat tcc atc cgc gcacga gtt gcg 1316 His Trp His Arg Trp Gln Asp His His Ser Ile Arg Ala ArgVal Ala 390 395 400 405 aga gcg atg tcc att ccc acc tca cct tcg cgc ctcagc ttc cac agc 1364 Arg Ala Met Ser Ile Pro Thr Ser Pro Ser Arg Leu SerPhe His Ser 410 415 420 atc aaa cag tcc acc tct gtc tga tacagggacgcataacattg aaaaagaaag 1418 Ile Lys Gln Ser Thr Ser Val 425 aggcaggcactctatcagtg agga 1442 36 428 PRT Ameiurus nebulosus 36 Met Trp Ile CysLeu Met Leu Lys Val Leu Ser Ile Leu Ser Phe Val 1 5 10 15 Val Val LysVal Ser Ala Asp Leu Thr Cys Asp Ala Val Leu Met Leu 20 25 30 Ala Ser GlyAsn His Thr Leu Tyr His Leu Asp Ala Ala Asn His Ser 35 40 45 Asp Thr AsnAsn Ser Gly Val Phe Cys Ser Thr Val Ile Asp Gly Ile 50 55 60 Gly Thr CysTrp Pro Arg Ser Val Ala Gly Glu Met Val Ser Arg Pro 65 70 75 80 Cys ProGlu Phe Leu Tyr Gly Val Arg Tyr Asn Thr Thr Asn Lys Ile 85 90 95 Phe ArgLys Cys Leu Ala Asn Gly Thr Trp Ala Pro Lys Ser Asn Tyr 100 105 110 SerGln Cys Lys Ala Ile Leu Asn Val Gln Arg Lys Ser Lys Leu His 115 120 125Tyr Arg Ile Ala Val Ile Ile Asn Tyr Leu Gly His Cys Leu Ser Leu 130 135140 Phe Thr Leu Leu Ile Ala Phe Ile Ile Phe Leu Arg Leu Arg Ser Ile 145150 155 160 Arg Cys Leu Arg Asn Ile Ile His Trp Asn Leu Thr Ser Ala PheIle 165 170 175 Leu Arg Asn Ala Thr Trp Phe Ile Val Gln Leu Thr Met AsnPro Asp 180 185 190 Val His Glu Ser Asn Val Pro Trp Cys Arg Leu Val ThrThr Ala Tyr 195 200 205 Asn Tyr Phe His Met Ala Asn Phe Phe Trp Met PheGly Glu Gly Cys 210 215 220 Tyr Leu His Thr Ala Ile Val Leu Thr Tyr SerThr Asp Lys Leu Lys 225 230 235 240 Lys Trp Met Phe Ile Cys Ile Gly TrpCys Ile Pro Ser Pro Ile Ile 245 250 255 Val Ala Trp Ala Ile Gly Lys LeuTyr Tyr Asp Asn Glu Lys Cys Trp 260 265 270 Phe Gly Lys Arg Ala Gly IleTyr Thr Asp Tyr Ile Tyr Gln Gly Pro 275 280 285 Met Ile Leu Val Leu MetIle Asn Phe Val Phe Leu Phe Asn Ile Val 290 295 300 Arg Ile Leu Met ThrLys Leu Arg Ala Ser Thr Thr Ser Glu Thr Ile 305 310 315 320 Gln Tyr ArgLys Ala Val Lys Ala Thr Leu Val Leu Leu Pro Leu Leu 325 330 335 Gly IleThr Tyr Met Leu Phe Phe Val Asn Pro Gly Glu Asp Glu Ile 340 345 350 SerGln Ile Val Phe Ile Tyr Phe Asn Ser Phe Leu Gln Ser Phe Gln 355 360 365Gly Phe Phe Val Ser Val Phe Tyr Cys Phe Leu Asn Ser Glu Val Arg 370 375380 Ser Ala Val Arg Lys His Trp His Arg Trp Gln Asp His His Ser Ile 385390 395 400 Arg Ala Arg Val Ala Arg Ala Met Ser Ile Pro Thr Ser Pro SerArg 405 410 415 Leu Ser Phe His Ser Ile Lys Gln Ser Thr Ser Val 420 42537 1218 DNA Ameiurus nebulosus CDS (1)..(1218) 37 atg gag gtc agt ctgctg gag ctg ctc agt gtg gag gtg aac tgc agc 48 Met Glu Val Ser Leu LeuGlu Leu Leu Ser Val Glu Val Asn Cys Ser 1 5 10 15 ctc gcg gac gcg tttgga gac cct gcg tac gga aac gca tca gac gct 96 Leu Ala Asp Ala Phe GlyAsp Pro Ala Tyr Gly Asn Ala Ser Asp Ala 20 25 30 ctg tac tgc aac gcc acggcg gat gag atc ggc acg tgc tgg ccg agg 144 Leu Tyr Cys Asn Ala Thr AlaAsp Glu Ile Gly Thr Cys Trp Pro Arg 35 40 45 agc ggc gcg ggg aga gtg gtggcg cgg ccg tgc ccc gac ttc atc aac 192 Ser Gly Ala Gly Arg Val Val AlaArg Pro Cys Pro Asp Phe Ile Asn 50 55 60 ggg gtc aag tac aac agc acc aggagc gcg tat aga gaa tgc ctg gag 240 Gly Val Lys Tyr Asn Ser Thr Arg SerAla Tyr Arg Glu Cys Leu Glu 65 70 75 80 aac ggc aca tgg gct ttc aag atcaac tac tcc agc tgc gag ccc att 288 Asn Gly Thr Trp Ala Phe Lys Ile AsnTyr Ser Ser Cys Glu Pro Ile 85 90 95 tta gag gaa aag agg aag tac ccg gtccac tac aag atc gct ctc atc 336 Leu Glu Glu Lys Arg Lys Tyr Pro Val HisTyr Lys Ile Ala Leu Ile 100 105 110 atc aac tat ttg gga cac tgc ata tctgta ggt gct ctc gtc atc gcc 384 Ile Asn Tyr Leu Gly His Cys Ile Ser ValGly Ala Leu Val Ile Ala 115 120 125 ttc gtt ctc ttc ctg tgc ttg aga agcatc cgg tgt ttg cgg aat gta 432 Phe Val Leu Phe Leu Cys Leu Arg Ser IleArg Cys Leu Arg Asn Val 130 135 140 att cac tgg aat tta ata acc acc ttcatc ctg agg aac atc atg tgg 480 Ile His Trp Asn Leu Ile Thr Thr Phe IleLeu Arg Asn Ile Met Trp 145 150 155 160 ctt ctg ctg cag ctc atc gac cacaac atc cat gaa agg aac gag ccg 528 Leu Leu Leu Gln Leu Ile Asp His AsnIle His Glu Arg Asn Glu Pro 165 170 175 tgg tgc cgc ctc ata acc acc gtctat aac tat ttt gtg gtg acg aat 576 Trp Cys Arg Leu Ile Thr Thr Val TyrAsn Tyr Phe Val Val Thr Asn 180 185 190 ttt ttc tgg atg ttc gtg gag ggctgt tat ctt cac aca gcc atc gtt 624 Phe Phe Trp Met Phe Val Glu Gly CysTyr Leu His Thr Ala Ile Val 195 200 205 atg acc tac tcc acc gac aag ctccgg aag tgg gtc ttc ctc ttc atc 672 Met Thr Tyr Ser Thr Asp Lys Leu ArgLys Trp Val Phe Leu Phe Ile 210 215 220 ggg tgg tgt att ccg tgt ccg gtcatc att gcg tgg gcc gtc ggg aag 720 Gly Trp Cys Ile Pro Cys Pro Val IleIle Ala Trp Ala Val Gly Lys 225 230 235 240 ctg tac aac gaa aac gaa cagtgc tgg ttt gga aaa gaa ccc gga aaa 768 Leu Tyr Asn Glu Asn Glu Gln CysTrp Phe Gly Lys Glu Pro Gly Lys 245 250 255 tac gtg gac tac att tat cagggt cct gtg att gtt gtt ctg ctg ata 816 Tyr Val Asp Tyr Ile Tyr Gln GlyPro Val Ile Val Val Leu Leu Ile 260 265 270 aac ttc gtg ttc ctg ttc aacatc gta cgt att ctc atg acg aag ctg 864 Asn Phe Val Phe Leu Phe Asn IleVal Arg Ile Leu Met Thr Lys Leu 275 280 285 cga gcc tcc acc acg tca gagacc ata cag tac agg aaa gcg gtg aag 912 Arg Ala Ser Thr Thr Ser Glu ThrIle Gln Tyr Arg Lys Ala Val Lys 290 295 300 gcg acg tta gtg ctg ctt cctctg ctc ggc atc aca tac atg ctg ttc 960 Ala Thr Leu Val Leu Leu Pro LeuLeu Gly Ile Thr Tyr Met Leu Phe 305 310 315 320 ttc gtg aat ccg ggg gatgat gac atc tca cag att gtc ttt att tat 1008 Phe Val Asn Pro Gly Asp AspAsp Ile Ser Gln Ile Val Phe Ile Tyr 325 330 335 ttc aat tcc ttc ctg cagtcc ttt cag ggt ttc ttc gtc tca gtg ttt 1056 Phe Asn Ser Phe Leu Gln SerPhe Gln Gly Phe Phe Val Ser Val Phe 340 345 350 tac tgc ttc ctc aac ggtgag gta cgg tca gca gta agg aaa cgt tgg 1104 Tyr Cys Phe Leu Asn Gly GluVal Arg Ser Ala Val Arg Lys Arg Trp 355 360 365 cac aga tgg cag gat aaccac gct ctc cgt gtt cgg gtt gcc agg gcg 1152 His Arg Trp Gln Asp Asn HisAla Leu Arg Val Arg Val Ala Arg Ala 370 375 380 atg tcc atc cca aca tcacct act cgc atc agc ttc cac agc att aaa 1200 Met Ser Ile Pro Thr Ser ProThr Arg Ile Ser Phe His Ser Ile Lys 385 390 395 400 cac acc acc gct gtgtga 1218 His Thr Thr Ala Val 405 38 405 PRT Ameiurus nebulosus 38 MetGlu Val Ser Leu Leu Glu Leu Leu Ser Val Glu Val Asn Cys Ser 1 5 10 15Leu Ala Asp Ala Phe Gly Asp Pro Ala Tyr Gly Asn Ala Ser Asp Ala 20 25 30Leu Tyr Cys Asn Ala Thr Ala Asp Glu Ile Gly Thr Cys Trp Pro Arg 35 40 45Ser Gly Ala Gly Arg Val Val Ala Arg Pro Cys Pro Asp Phe Ile Asn 50 55 60Gly Val Lys Tyr Asn Ser Thr Arg Ser Ala Tyr Arg Glu Cys Leu Glu 65 70 7580 Asn Gly Thr Trp Ala Phe Lys Ile Asn Tyr Ser Ser Cys Glu Pro Ile 85 9095 Leu Glu Glu Lys Arg Lys Tyr Pro Val His Tyr Lys Ile Ala Leu Ile 100105 110 Ile Asn Tyr Leu Gly His Cys Ile Ser Val Gly Ala Leu Val Ile Ala115 120 125 Phe Val Leu Phe Leu Cys Leu Arg Ser Ile Arg Cys Leu Arg AsnVal 130 135 140 Ile His Trp Asn Leu Ile Thr Thr Phe Ile Leu Arg Asn IleMet Trp 145 150 155 160 Leu Leu Leu Gln Leu Ile Asp His Asn Ile His GluArg Asn Glu Pro 165 170 175 Trp Cys Arg Leu Ile Thr Thr Val Tyr Asn TyrPhe Val Val Thr Asn 180 185 190 Phe Phe Trp Met Phe Val Glu Gly Cys TyrLeu His Thr Ala Ile Val 195 200 205 Met Thr Tyr Ser Thr Asp Lys Leu ArgLys Trp Val Phe Leu Phe Ile 210 215 220 Gly Trp Cys Ile Pro Cys Pro ValIle Ile Ala Trp Ala Val Gly Lys 225 230 235 240 Leu Tyr Asn Glu Asn GluGln Cys Trp Phe Gly Lys Glu Pro Gly Lys 245 250 255 Tyr Val Asp Tyr IleTyr Gln Gly Pro Val Ile Val Val Leu Leu Ile 260 265 270 Asn Phe Val PheLeu Phe Asn Ile Val Arg Ile Leu Met Thr Lys Leu 275 280 285 Arg Ala SerThr Thr Ser Glu Thr Ile Gln Tyr Arg Lys Ala Val Lys 290 295 300 Ala ThrLeu Val Leu Leu Pro Leu Leu Gly Ile Thr Tyr Met Leu Phe 305 310 315 320Phe Val Asn Pro Gly Asp Asp Asp Ile Ser Gln Ile Val Phe Ile Tyr 325 330335 Phe Asn Ser Phe Leu Gln Ser Phe Gln Gly Phe Phe Val Ser Val Phe 340345 350 Tyr Cys Phe Leu Asn Gly Glu Val Arg Ser Ala Val Arg Lys Arg Trp355 360 365 His Arg Trp Gln Asp Asn His Ala Leu Arg Val Arg Val Ala ArgAla 370 375 380 Met Ser Ile Pro Thr Ser Pro Thr Arg Ile Ser Phe His SerIle Lys 385 390 395 400 His Thr Thr Ala Val 405 39 1248 DNA Bos taurusCDS (1)..(1248) 39 atg gga cgg cgc ccg cag ctc cgg ctt gtc aag gcc cttctc ctc ctg 48 Met Gly Arg Arg Pro Gln Leu Arg Leu Val Lys Ala Leu LeuLeu Leu 1 5 10 15 ggg ctg aac tcc atc tct gcc tcc ctc cag gac cag cattgc gag agc 96 Gly Leu Asn Ser Ile Ser Ala Ser Leu Gln Asp Gln His CysGlu Ser 20 25 30 ttg tcc gtg gcc agc aac gtc tct gga ctg cag tgc aat gcttcc gtg 144 Leu Ser Val Ala Ser Asn Val Ser Gly Leu Gln Cys Asn Ala SerVal 35 40 45 gac ctt att ggt acc tgc tgg ccc cag agt cct gca ggg cag ttggtg 192 Asp Leu Ile Gly Thr Cys Trp Pro Gln Ser Pro Ala Gly Gln Leu Val50 55 60 gtt cga ccc tgc ctc gta ttt ttc tat ggt gtc cgc tac aat acc aca240 Val Arg Pro Cys Leu Val Phe Phe Tyr Gly Val Arg Tyr Asn Thr Thr 6570 75 80 aac aac ggc tac cgg gag tgc ctg gcc aat ggc acg tgg gcc gcc cgc288 Asn Asn Gly Tyr Arg Glu Cys Leu Ala Asn Gly Thr Trp Ala Ala Arg 8590 95 gtg aac tac tcc gag tgc caa gag atc ctc agc gag gag aag aag agc336 Val Asn Tyr Ser Glu Cys Gln Glu Ile Leu Ser Glu Glu Lys Lys Ser 100105 110 aag gtg cac tac cac atc gct gtc atc atc aac tac cta ggc cac tgc384 Lys Val His Tyr His Ile Ala Val Ile Ile Asn Tyr Leu Gly His Cys 115120 125 atc tcc ctg gcg gcc ctc ctg gtg gcc ttt gtc ctc ttt ctg cgg ctc432 Ile Ser Leu Ala Ala Leu Leu Val Ala Phe Val Leu Phe Leu Arg Leu 130135 140 agg agc atc cgg tgc ctg aga aac atc atc cac tgg aac ctc atc tca480 Arg Ser Ile Arg Cys Leu Arg Asn Ile Ile His Trp Asn Leu Ile Ser 145150 155 160 gcc ttc atc ctg cgc aat gcc acg tgg ttc gtg gtc cag ctc accatg 528 Ala Phe Ile Leu Arg Asn Ala Thr Trp Phe Val Val Gln Leu Thr Met165 170 175 agc ccc gaa gtc cat cag agc aac gtg ggc tgg tgc agg ctg gtgaca 576 Ser Pro Glu Val His Gln Ser Asn Val Gly Trp Cys Arg Leu Val Thr180 185 190 gcc gcc tac aac tac ttc cac gtg acc aac ttc ttc tgg atg ttcggt 624 Ala Ala Tyr Asn Tyr Phe His Val Thr Asn Phe Phe Trp Met Phe Gly195 200 205 gag ggc tgc tac ctg cac acg gcc atc gtg ctc acg tac tcc acagac 672 Glu Gly Cys Tyr Leu His Thr Ala Ile Val Leu Thr Tyr Ser Thr Asp210 215 220 cgg ctg cga aag tgg atg ttt atc tgc atc ggc tgg ggt gtg cctttc 720 Arg Leu Arg Lys Trp Met Phe Ile Cys Ile Gly Trp Gly Val Pro Phe225 230 235 240 ccc atc att gtg gcc tgg gcc att ggg aag ctg tac tac gacaat gag 768 Pro Ile Ile Val Ala Trp Ala Ile Gly Lys Leu Tyr Tyr Asp AsnGlu 245 250 255 aag tgc tgg ttt ggc aaa agg cct ggg gtg tac act gac tacatc tac 816 Lys Cys Trp Phe Gly Lys Arg Pro Gly Val Tyr Thr Asp Tyr IleTyr 260 265 270 cag ggc ccg atg atc ttg gtc ctg ctg atc aat ttc atc ttcctt ttc 864 Gln Gly Pro Met Ile Leu Val Leu Leu Ile Asn Phe Ile Phe LeuPhe 275 280 285 aac atc gtc cgc atc ctc atg acc aaa ctc cgg gca tcc accacc tct 912 Asn Ile Val Arg Ile Leu Met Thr Lys Leu Arg Ala Ser Thr ThrSer 290 295 300 gag acc att cag tac agg aag gct gtg aag gcc act ctg gtgctg ctc 960 Glu Thr Ile Gln Tyr Arg Lys Ala Val Lys Ala Thr Leu Val LeuLeu 305 310 315 320 ccc ctc ctg ggc atc acg tac atg ctg ttc ttc gtg aaccct ggg gag 1008 Pro Leu Leu Gly Ile Thr Tyr Met Leu Phe Phe Val Asn ProGly Glu 325 330 335 gac gag gtc tcc agg gtc gtc ttc atc tac ttc aac tccttc ctg gaa 1056 Asp Glu Val Ser Arg Val Val Phe Ile Tyr Phe Asn Ser PheLeu Glu 340 345 350 tct ttc cag ggc ttc ttc gtg tct gtg ttc tac tgc ttcctc aac agc 1104 Ser Phe Gln Gly Phe Phe Val Ser Val Phe Tyr Cys Phe LeuAsn Ser 355 360 365 gag gtc cgc tct gcc atc cgg aag agg tgg cac cgc tggcag gac aag 1152 Glu Val Arg Ser Ala Ile Arg Lys Arg Trp His Arg Trp GlnAsp Lys 370 375 380 cac tca atc cgt gcc cgc gtg gct cgc gcc atg tcc atcccc acc tcc 1200 His Ser Ile Arg Ala Arg Val Ala Arg Ala Met Ser Ile ProThr Ser 385 390 395 400 ccc acc cgt gtc agc ttt cac agc atc aag cag tccaca gca gtg tga 1248 Pro Thr Arg Val Ser Phe His Ser Ile Lys Gln Ser ThrAla Val 405 410 415 40 415 PRT Bos taurus 40 Met Gly Arg Arg Pro Gln LeuArg Leu Val Lys Ala Leu Leu Leu Leu 1 5 10 15 Gly Leu Asn Ser Ile SerAla Ser Leu Gln Asp Gln His Cys Glu Ser 20 25 30 Leu Ser Val Ala Ser AsnVal Ser Gly Leu Gln Cys Asn Ala Ser Val 35 40 45 Asp Leu Ile Gly Thr CysTrp Pro Gln Ser Pro Ala Gly Gln Leu Val 50 55 60 Val Arg Pro Cys Leu ValPhe Phe Tyr Gly Val Arg Tyr Asn Thr Thr 65 70 75 80 Asn Asn Gly Tyr ArgGlu Cys Leu Ala Asn Gly Thr Trp Ala Ala Arg 85 90 95 Val Asn Tyr Ser GluCys Gln Glu Ile Leu Ser Glu Glu Lys Lys Ser 100 105 110 Lys Val His TyrHis Ile Ala Val Ile Ile Asn Tyr Leu Gly His Cys 115 120 125 Ile Ser LeuAla Ala Leu Leu Val Ala Phe Val Leu Phe Leu Arg Leu 130 135 140 Arg SerIle Arg Cys Leu Arg Asn Ile Ile His Trp Asn Leu Ile Ser 145 150 155 160Ala Phe Ile Leu Arg Asn Ala Thr Trp Phe Val Val Gln Leu Thr Met 165 170175 Ser Pro Glu Val His Gln Ser Asn Val Gly Trp Cys Arg Leu Val Thr 180185 190 Ala Ala Tyr Asn Tyr Phe His Val Thr Asn Phe Phe Trp Met Phe Gly195 200 205 Glu Gly Cys Tyr Leu His Thr Ala Ile Val Leu Thr Tyr Ser ThrAsp 210 215 220 Arg Leu Arg Lys Trp Met Phe Ile Cys Ile Gly Trp Gly ValPro Phe 225 230 235 240 Pro Ile Ile Val Ala Trp Ala Ile Gly Lys Leu TyrTyr Asp Asn Glu 245 250 255 Lys Cys Trp Phe Gly Lys Arg Pro Gly Val TyrThr Asp Tyr Ile Tyr 260 265 270 Gln Gly Pro Met Ile Leu Val Leu Leu IleAsn Phe Ile Phe Leu Phe 275 280 285 Asn Ile Val Arg Ile Leu Met Thr LysLeu Arg Ala Ser Thr Thr Ser 290 295 300 Glu Thr Ile Gln Tyr Arg Lys AlaVal Lys Ala Thr Leu Val Leu Leu 305 310 315 320 Pro Leu Leu Gly Ile ThrTyr Met Leu Phe Phe Val Asn Pro Gly Glu 325 330 335 Asp Glu Val Ser ArgVal Val Phe Ile Tyr Phe Asn Ser Phe Leu Glu 340 345 350 Ser Phe Gln GlyPhe Phe Val Ser Val Phe Tyr Cys Phe Leu Asn Ser 355 360 365 Glu Val ArgSer Ala Ile Arg Lys Arg Trp His Arg Trp Gln Asp Lys 370 375 380 His SerIle Arg Ala Arg Val Ala Arg Ala Met Ser Ile Pro Thr Ser 385 390 395 400Pro Thr Arg Val Ser Phe His Ser Ile Lys Gln Ser Thr Ala Val 405 410 41541 1422 DNA Gallus gallus CDS (138)..(1400) 41 tcacagggag gttataaaaggcagtgagtg gggagcgggg cacgcggagc cacctgagca 60 cgaggatttg gagccccgacggcagcggga gcggagccgg ccatgccccg ggtcgttggg 120 tgcggaggga gctaagg atggtg ccc ggc ccg cgt cct gcc ctc ctc ctc 170 Met Val Pro Gly Pro Arg ProAla Leu Leu Leu 1 5 10 ctc ctc ttt ctc ctg cag gcg ttt ctc ctc tgg gatagt ccc gtt gca 218 Leu Leu Phe Leu Leu Gln Ala Phe Leu Leu Trp Asp SerPro Val Ala 15 20 25 gcc tcc atc caa gag cag tac tgt gag agc ctg ctg cccacc acc aac 266 Ala Ser Ile Gln Glu Gln Tyr Cys Glu Ser Leu Leu Pro ThrThr Asn 30 35 40 cac aca gga cct cag tgc aac gcc tcg gtg gac ctg att ggcacg tgc 314 His Thr Gly Pro Gln Cys Asn Ala Ser Val Asp Leu Ile Gly ThrCys 45 50 55 tgg ccc cgc agt gca gtg gga caa ctg gtg gct cgg ccc tgc cccgag 362 Trp Pro Arg Ser Ala Val Gly Gln Leu Val Ala Arg Pro Cys Pro Glu60 65 70 75 tat ttc tac ggc gtg cgg tac aac acc aca aac aat ggc tac agggaa 410 Tyr Phe Tyr Gly Val Arg Tyr Asn Thr Thr Asn Asn Gly Tyr Arg Glu80 85 90 tgc ctc gct aac ggg agc tgg gca gca cgg gtc aac tac tcc cag tgc458 Cys Leu Ala Asn Gly Ser Trp Ala Ala Arg Val Asn Tyr Ser Gln Cys 95100 105 cag gag atc ctc agt gag gag aag agg agc aag ctg cac tac cac atc506 Gln Glu Ile Leu Ser Glu Glu Lys Arg Ser Lys Leu His Tyr His Ile 110115 120 gct gtc atc atc aac tac ctg ggg cac tgc gtc tcg ctg ggg acc ctc554 Ala Val Ile Ile Asn Tyr Leu Gly His Cys Val Ser Leu Gly Thr Leu 125130 135 ctt gtg gcc ttc gtc ctc ttc atg cgc ctg cgg agc atc cgg tgc ttg602 Leu Val Ala Phe Val Leu Phe Met Arg Leu Arg Ser Ile Arg Cys Leu 140145 150 155 agg aac atc atc cac tgg aac ctg atc aca gcc ttc atc cta cgcaat 650 Arg Asn Ile Ile His Trp Asn Leu Ile Thr Ala Phe Ile Leu Arg Asn160 165 170 gcc acg tgg ttt gtg gtg cag ctc acg atg aac cca gag gtg cacgag 698 Ala Thr Trp Phe Val Val Gln Leu Thr Met Asn Pro Glu Val His Glu175 180 185 agc aac gtg gtc tgg tgc cgc ttg gtc act gct gcc tac aat tacttc 746 Ser Asn Val Val Trp Cys Arg Leu Val Thr Ala Ala Tyr Asn Tyr Phe190 195 200 cat gtc acc aac ttc ttc tgg atg ttt ggc gag ggc tgc tac ctgcac 794 His Val Thr Asn Phe Phe Trp Met Phe Gly Glu Gly Cys Tyr Leu His205 210 215 aca gcc atc gtc ctc acc tat tcc acc gac aag ctc cgc aag tggatg 842 Thr Ala Ile Val Leu Thr Tyr Ser Thr Asp Lys Leu Arg Lys Trp Met220 225 230 235 ttc atc tgc att ggc tgg tgt atc ccc ttt ccc atc att gtcgcc tgg 890 Phe Ile Cys Ile Gly Trp Cys Ile Pro Phe Pro Ile Ile Val AlaTrp 240 245 250 gcc atc ggg aag ctg tac tac gac aac gag aag tgc tgg tttggg aag 938 Ala Ile Gly Lys Leu Tyr Tyr Asp Asn Glu Lys Cys Trp Phe GlyLys 255 260 265 cga gca gga gtt tat act gac tac atc tat caa ggt ccc atgatc ctg 986 Arg Ala Gly Val Tyr Thr Asp Tyr Ile Tyr Gln Gly Pro Met IleLeu 270 275 280 gtg ctt ctg atc aac ttc atc ttt ctg ttc aac att gtt cggatt ctc 1034 Val Leu Leu Ile Asn Phe Ile Phe Leu Phe Asn Ile Val Arg IleLeu 285 290 295 atg acc aag ctc cga gca tca acc acg tca gag aca atc cagtac aga 1082 Met Thr Lys Leu Arg Ala Ser Thr Thr Ser Glu Thr Ile Gln TyrArg 300 305 310 315 aaa gca gtc aag gct acg ctg gtg ctg ctg tcc ttg ctggga atc acc 1130 Lys Ala Val Lys Ala Thr Leu Val Leu Leu Ser Leu Leu GlyIle Thr 320 325 330 tac atg ctg ttc ttt gtc aat ccg ggg gag gat gag atctcc agg atc 1178 Tyr Met Leu Phe Phe Val Asn Pro Gly Glu Asp Glu Ile SerArg Ile 335 340 345 gtc ttc atc tac ttc aac tcc ttc ctg gag tcc ttc cagggc ttc ttt 1226 Val Phe Ile Tyr Phe Asn Ser Phe Leu Glu Ser Phe Gln GlyPhe Phe 350 355 360 gtc tct gtc ttc tac tgc ttc ctg aac agc gag gtg cgttcg gct gtg 1274 Val Ser Val Phe Tyr Cys Phe Leu Asn Ser Glu Val Arg SerAla Val 365 370 375 cgg aag cgg tgg cac cga tgg cag gac aag cac tcc atccgc gct cgg 1322 Arg Lys Arg Trp His Arg Trp Gln Asp Lys His Ser Ile ArgAla Arg 380 385 390 395 gtg gct cgg gcc atg tcc atc ccc acc tcc cca acccgg gtc agc ttc 1370 Val Ala Arg Ala Met Ser Ile Pro Thr Ser Pro Thr ArgVal Ser Phe 400 405 410 cac agc atc aag cag tcc tca gca gtg tgaggcaggagga ggcagctgcc ga 1422 His Ser Ile Lys Gln Ser Ser Ala Val 415420 42 420 PRT Gallus gallus 42 Met Val Pro Gly Pro Arg Pro Ala Leu LeuLeu Leu Leu Phe Leu Leu 1 5 10 15 Gln Ala Phe Leu Leu Trp Asp Ser ProVal Ala Ala Ser Ile Gln Glu 20 25 30 Gln Tyr Cys Glu Ser Leu Leu Pro ThrThr Asn His Thr Gly Pro Gln 35 40 45 Cys Asn Ala Ser Val Asp Leu Ile GlyThr Cys Trp Pro Arg Ser Ala 50 55 60 Val Gly Gln Leu Val Ala Arg Pro CysPro Glu Tyr Phe Tyr Gly Val 65 70 75 80 Arg Tyr Asn Thr Thr Asn Asn GlyTyr Arg Glu Cys Leu Ala Asn Gly 85 90 95 Ser Trp Ala Ala Arg Val Asn TyrSer Gln Cys Gln Glu Ile Leu Ser 100 105 110 Glu Glu Lys Arg Ser Lys LeuHis Tyr His Ile Ala Val Ile Ile Asn 115 120 125 Tyr Leu Gly His Cys ValSer Leu Gly Thr Leu Leu Val Ala Phe Val 130 135 140 Leu Phe Met Arg LeuArg Ser Ile Arg Cys Leu Arg Asn Ile Ile His 145 150 155 160 Trp Asn LeuIle Thr Ala Phe Ile Leu Arg Asn Ala Thr Trp Phe Val 165 170 175 Val GlnLeu Thr Met Asn Pro Glu Val His Glu Ser Asn Val Val Trp 180 185 190 CysArg Leu Val Thr Ala Ala Tyr Asn Tyr Phe His Val Thr Asn Phe 195 200 205Phe Trp Met Phe Gly Glu Gly Cys Tyr Leu His Thr Ala Ile Val Leu 210 215220 Thr Tyr Ser Thr Asp Lys Leu Arg Lys Trp Met Phe Ile Cys Ile Gly 225230 235 240 Trp Cys Ile Pro Phe Pro Ile Ile Val Ala Trp Ala Ile Gly LysLeu 245 250 255 Tyr Tyr Asp Asn Glu Lys Cys Trp Phe Gly Lys Arg Ala GlyVal Tyr 260 265 270 Thr Asp Tyr Ile Tyr Gln Gly Pro Met Ile Leu Val LeuLeu Ile Asn 275 280 285 Phe Ile Phe Leu Phe Asn Ile Val Arg Ile Leu MetThr Lys Leu Arg 290 295 300 Ala Ser Thr Thr Ser Glu Thr Ile Gln Tyr ArgLys Ala Val Lys Ala 305 310 315 320 Thr Leu Val Leu Leu Ser Leu Leu GlyIle Thr Tyr Met Leu Phe Phe 325 330 335 Val Asn Pro Gly Glu Asp Glu IleSer Arg Ile Val Phe Ile Tyr Phe 340 345 350 Asn Ser Phe Leu Glu Ser PheGln Gly Phe Phe Val Ser Val Phe Tyr 355 360 365 Cys Phe Leu Asn Ser GluVal Arg Ser Ala Val Arg Lys Arg Trp His 370 375 380 Arg Trp Gln Asp LysHis Ser Ile Arg Ala Arg Val Ala Arg Ala Met 385 390 395 400 Ser Ile ProThr Ser Pro Thr Arg Val Ser Phe His Ser Ile Lys Gln 405 410 415 Ser SerAla Val 420 43 38 PRT Mus musculus 43 Val Ile Leu Ser Leu Asp Val ProIle Gly Leu Leu Arg Ile Leu Leu 1 5 10 15 Glu Gln Ala Arg Tyr Lys AlaAla Arg Asn Gln Ala Ala Thr Asn Ala 20 25 30 Gln Ile Leu Ala His Val 3544 38 PRT Homo sapiens 44 Ile Val Leu Ser Leu Asp Val Pro Ile Gly LeuLeu Gln Ile Leu Leu 1 5 10 15 Glu Gln Ala Arg Ala Arg Ala Ala Arg GluGln Ala Thr Thr Asn Ala 20 25 30 Arg Ile Leu Ala Arg Val 35

What is claimed is:
 1. A method for identifying candidate compounds forregulating skeletal muscle mass or function, comprising: (a) contactinga test compound with a CRF₂R; (b) determining whether the test compoundbinds to the CRF₂ R; and (c) identifying those test compounds which bindto the CRF₂R as candidate compounds for regulating skeletal muscle massor function.
 2. A method for identifying candidate compounds forregulating skeletal muscle mass or function, comprising: (a) contactinga test compound with a cell expressing a functional CRF₂R, (b)determining whether the test compound activates the CRF₂R; and (c)identifying those test compounds which activate the CRF₂R as candidatecompounds for regulating skeletal muscle mass or function.
 3. The methodfor identifying candidate compounds according to claim 2, the methodfurther comprising generating a list of candidate compounds.
 4. Themethod for identifying candidate compounds according to claim 2, inwhich the CRF₂R is expressed on a eukaryotic cell.
 5. The method foridentifying candidate compounds according to claim 4 wherein the CRF₂Rhas an amino acid sequence that is greater than 80% identical to thesequence of SEQ ID NO:
 10. 6. The method for identifying candidatecompounds according to claim 4 wherein the CRF₂R has an amino acidsequence that is greater than 90% identical to the sequence of SEQ IDNO:
 10. 7. The method for identifying candidate compounds according toclaim 4 wherein the CRF₂R has the amino acid sequence corresponding tothe amino acid sequence of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQID NO:18, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:32 or SEQID NO:38.
 8. The method for identifying candidate compounds according toclaim 4, wherein the CRF₂R is from a species selected from the groupconsisting of human, mouse and rat.
 9. The method for identifyingcandidate compounds according to claim 8, wherein the CRF₂R is human.10. The method for identifying candidate compounds according to claim 4,the cell having a cellular cAMP level, in which determining whether thetest compound activates the CRF₂R involves measuring the cellular cAMPlevel.
 11. The method for identifying candidate compounds according toclaim 10, in which the cell further comprises a reporter geneoperatively associated with a cAMP responsive element and measuring thecellular cAMP level involves measuring expression of the reporter gene.12. The method for identifying candidate compounds according to claim11, in which the reporter gene is alkaline phosphatase, chloramphenicolacetyltransferase, luciferase, glucuronide synthetase, growth hormone,placental alkaline phosphatase or Green Fluorescent Protein.
 13. Amethod for identifying candidate compounds for regulating skeletalmuscle mass or function comprising, in any order: (a) contacting a testcompound with a cell expressing a functional CRF₂R, and determining alevel of activation of CRF₂R resulting from the test compound; (b)contacting a test compound with a cell expressing a functional CRF₁R,and determining the level of activation of CRF₁R resulting from the testcompound; followed by (c) comparing the level of CRF₂R activation andthe level of CRF₁R activation; and (d) identifying those test compoundsthat show similar activity toward CRF₂R and CRF₁R or show selectivityfor CRF₂R as candidate compounds for regulating skeletal muscle mass orfunction.
 14. The method according to claim 13 wherein the candidatecompound exhibits a 100-fold or greater selectivity for CRF₂R.
 15. Themethod according to claim 14 wherein the candidate compound exhibits a1000-fold or greater selectivity for CRF₂R.
 16. The method according toclaim 13 wherein the candidate compound exhibits between 1-fold and100-fold selectivity for CRF₂R.
 17. A method for identifying candidatetherapeutic compounds from a group of one or more candidate compoundswhich have been previously determined to bind to or activate the CRF₂Rcomprising: (a) administering the candidate compound to a non-humananimal; and (b) determining whether the candidate compound regulatesskeletal muscle mass or function in the treated animal.
 18. A method foridentifying candidate compounds that prolong or augment theagonist-induced activation of a CRF₂R or of a CRF₂R signal transductionpathway, comprising, in any order or concurrently: (a) contacting a testcompound with a cell which expresses a functional CRF₂R; (b) treatingthe cell with a CRF₂R agonist for a sufficient time and at a sufficientconcentration to cause desensitization of the CRF₂R in control cells;followed by (c) determining the level of activation of the CRF₂R; and(d) identifying those test compounds that prolong or augment theactivation of a CRF₂R or of a CRF₂R signal transduction pathway ascandidate compounds for regulating skeletal muscle mass or function. 19.A method for identifying candidate therapeutic compounds from a group ofone or more candidate compounds which have been previously determined toprolong or augment the activation of a CRF₂R or of a CRF₂R signaltransduction pathway comprising: a) administering the candidatecompound, in conjunction with a CRF₂R agonist, to a non-human animal;and b) determining whether the candidate compound regulates skeletalmuscle mass or function in the treated animal.
 20. A method foridentifying candidate compounds for increasing CRF₂R or CFR expression,comprising: (a) contacting a test compound with a cell or cell lysatecontaining a reporter gene operatively associated with a CRF₂Rregulatory element or a CRF regulatory element; (b) detecting expressionof the reporter gene; and (c) identifying those test compounds thatincrease expression of the reporter gene as candidate compounds forregulating skeletal muscle mass or function.
 21. A method for increasingskeletal muscle mass or function in a subject in which such an increaseis desirable, comprising: (a) identifying a subject in which an increasein muscle mass or function is desirable; and (b) administering to thesubject a safe and effective amount of a CRF₂R agonist.
 22. A method fortreating skeletal muscle atrophy in a subject in need of such treatment,comprising: (a) identifying a subject in need of treatment for skeletalmuscle atrophy; and (b) administering to the subject a safe andeffective amount of a compound selected from the group consisting of aCRF₂R agonist, an expression vector encoding a functional CRF₂R, anexpression vector encoding a constitutively active CRF₂R, an expressionvector encoding a CRF and a compound that increases expression of CRF₂Ror CRF.
 23. A method for treating skeletal muscle atrophy according toclaim 22 wherein the compound is a CRF₂R agonist.
 24. The method fortreating skeletal muscle atrophy according to claim 23, wherein theCRF₂R agonist is identified by a method for identifying candidatecompounds for regulating skeletal muscle mass or function, comprising:(a) contacting a test compound with a CRF₂R, (b) determining whether thetest compound activates the CRF₂R; and (c) identifying those testcompounds that activate the CRF₂R as candidate compounds for regulatingskeletal muscle mass or function.
 25. The method for treating skeletalmuscle atrophy according to claim 23, wherein the CRF₂R agonist is achimeric or human antibody.
 26. The method according to claim 25 whereinthe CRF₂R agonist is selective for CRF₂R over CRF₁R.
 27. The methodaccording to claim 26 wherein the CRF₂R agonist exhibits selectivity forCRF₂R greater that or equal to 100-fold.
 28. The method according toclaim 23 wherein the subject has muscular dystrophy and the CRF₂Ragonist exhibits between 1-fold and 100-fold selectivity for CRF₂R. 29.The method for treating skeletal muscle atrophy according to claim 23,further comprising: administering to the subject a safe and effectiveamount of a compound that prolongs or augments the activation of CRF₂Ror the activation of a CRF₂R signal transduction pathway.
 30. A purifiedantibody specific for a CRF₂R, wherein the antibody is a chimeric orhuman antibody.
 31. The antibody of claim 30, wherein the antibody is anagonist of a CRF₂R.
 32. The antibody of claim 31, wherein the antibodyis a human antibody.
 33. A pharmaceutical composition, comprising: (a) asafe and effective amount of a CRF₂R agonist; and (b) apharmaceutically-acceptable carrier.
 34. The pharmaceutical compositionaccording to claim 33 wherein the CRF₂R agonist is a chimeric or humanantibody specific for a CRF₂R.
 35. The pharmaceutical compositionaccording to claim 33 wherein the CRF₂R agonist is urocortin II.